Receptor Signal Transduction Protocols

1 Radioligand-Binding Methods for Membrane Preparations and Intact Cells Mary Keen 1. Introduction Radiollgand binding is a straightforward technique that measures the bindmg of a labeled agonist or antagonist to its receptor. It is applicable to a variety of receptor preparations, ranging from purified receptors to tissue slices, or even whole animals. However, membranes or broken-cell preparations are undoubtedly the most widely used. Radioligand binding allows the affinity of drugs for their receptors to be determined very readily and It also allows the number of receptors in a tissue or cell to be quantified-something that was impossible before the mtroductlon of binding techniques. Furthermore, the technique can be adapted to study the association and dissociation kinetics of llgand binding (1,2), as well as complex allosteric mteractlons between ligands (2) or between receptors and effector molecules, such as guanine nucleotidebinding proteins (G proteins) (2,2). Despite the ease with which radloligand binding can provide information regarding a wide range of receptors, it does have its limitations. There is an absolute requirement for a high-affinity radioligand, selective for the receptor of Interest. Even if such a radioligand exists, it may still be impossible to detect receptors in a particular tissue if receptor abundance is low relative to the nonspecific binding of the radioligand. As with any technique, radioligand-binding data can be beset with artifacts if experiments are not designed carefully. Most importantly, it must always be remembered that radioligand-binding experiments identify ligand-binding sites, which may or may not represent bonafide receptors. These points are discussed m more detail m refs. 1-3. This chapter provides detailed instructions on how to obtain and analyze equilibrium-binding data for the IP prostanoid (prostacyclin) receptor in human From

Methods m Molecular Bralogy, vol 83 Receptor S/gnal Transducbon Edlted by R A J Chalks Humana Press Inc. Totowa, NJ

1

Protocols

Keen

2

platelets and the neuroblastoma x glioma cell line, NGlO%15, using the labeled agonist, [3H]-iloprost, as a radioligand. This system illustrates many of the more common problems inherent in the design and analysis of radioligand binding experiments, because [3H]-iloprost is a rather difficult ligand. 2. Materials 1. Platelets: Bags of frozen time-expired human platelets may be obtained from the Blood Transfusion Service (London, UK) and stored at -70°C until required. 2. NG108-15 cells: Confluent NG108-15 cells (see Note 1) are harvested by agitation in phosphate buffered saline and the cells pelleted by a low-speed spin (200g for 2 mm). The pelleted cells may then be stored at -70°C until required. 3. Lysis buffer: 5 mM Tris-HCI, pH 7.4. 4. Wash buffer: 50 mMTris-HCl, 0.25 mM EDTA, pH 7.4. 5. Assay buffer: 50 mA4Tris-HCI, 5 mMMgCl,, pH 7.4 Note: Items 3-5 may all be prepared in liter quantrties and stored at 4°C until required 6. [3H]-iloprost may be obtained from Amersham International, Amersham, UK The stock should be stored at -20°C and diluted in assay buffer on the day of the experiment. 7. Iloprost: Unlabeled iloprost is supplied with [3H]-iloprost from Amersham International or Schermg AG, Berlin, Germany. The stock concentrations supplied may be stored at 4OC, being diluted m assay buffer on the day of the experiment. 8. Cell harvester and filters: A 24-place Brandel cell harvester and GF/B filters were used in the experiments presented here. Both cell harvester and filters may be obtained through Semat Technical, St. Albans, UK. 9. 5’-Guanylylimidodiphosphate (GppNHp [Sigma, Poole, UK]): 2.5 mMGppNHp (to give a final concentration of 100 pM) may be made up in I-mL quantities in assay buffer on the day of the experiment and stored on ice. Any excess may be frozen at -2O’C and used in a subsequent assay. 10. Curve-fitting programs: A wide range of suitable programs are available, but a detailed comparison is beyond the scope of this chapter. However, it is useful to choose a program that allows parameters to be constrained to particular values, user-defined equations to be entered, and publication-quality output to be produced, The analysis and figures reproduced here were obtained using FigP (Biosofi, Cambridge, UK).

3. Methods 3.1. Preparation

of Platelet Membranes

1. Thaw the bag of frozen platelets in cold water. Centrifuge at 80,OOOg for 35 mm

at 4°C. Discard the superuatant 2. Resuspendthe pellet in roughly 10 mL/U platelets of ice-cold 5 mA4Tris-HCl, pH 7.4. Lyse any intact platelets by either stirring the suspensionon ice for 30 min or freezing the suspension overnight and then thawing. Use the most convenient method.

Radioligand-Binding Methods

3

3. Centrifuge the lysed platelets at 80,OOOg for 30 min at 4’C. Discard the supematam and resuspend the pellet in a convenient volume of ice-cold wash buffer. 4. Repeat step 3 once. 5. Centrifuge at 80,OOOg for 30 min at 4°C. Resuspend the pellet in wash buffer to a concentration of about 20 mg protein/mL.‘As a rough guide, 1 mL of pelleted membranes is equivalent to about 100 mg protein. Freeze the final suspension in 1-mL aliquots at -70°C until required. 6. On the day of the binding assay, thaw the frozen membranes and dilute 1: 10 with assay buffer. Do this a few minutes before startmg the binding assay, so that the membrane suspension just has time to come up to room temperature before pipeting (see Note 2).

3.2. Preparation

of NG108-15 Cell Homogenates

1. On the day of the binding assay, thaw the pelleted NGl08- 15 cells and resuspend in assay buffer, using 20 strokes of a tight-fitting glass Dounce homogenizer (Jencons, Leighton Buzzard, UK) or something similar. The pellet from an 80-cm2 cell culture flask homogenized in about 7 mL assay buffer provides sufficient homogenate for a single binding curve in thrs system. Prepare the homogenate a few minutes before starting the bmding assay, so that the suspension has Just enough time to come up to room temperature before pipetmg (see Note 2). 2. NG108-15 cells may also be prepared as washed homogenates (see Note 3) or whole cells (see Note 4).

3.3. Direct (Saturation) Binding This type of assay measures the equilibrium binding of a range of concentrations of the radioligand. As no radioligand binds exclustvely to its receptor, it is also necessary to determine the nonspecific binding of each concentration of radioligand by suppressing its binding to the receptor by the inclusion of a saturating concentration of an unlabeled ligand (see Note 5). Specific binding can then be calculated as the difference between total and nonspecific binding (see Section 3.5.). 1. The design of the binding experiment depends largely on the method to be used

in separatingbound and free ligand (see Note 6). In the experimentspresented here, a Brandel cell harvester was used, which filters 24 samples simultaneously. Therefore, each binding curve was designed to use 24 samples; 6 concentrations of [3H]-iloprost (0.3-100 nM; see Notes 7 and 8), each prepared in the absence

and presence of 10 pM unlabeled iloprost, to define nonspecific binding (see Note 5), with eachdetermination being performed in duplicate. 2. Prepare appropriate dilutions of all ligands, and so on, in assay buffer (see Note 9). 3. Pipet the assay components into polypropylene assay tubes (see Note 10) as follows: 30 pL assay buffer, 10 pL [3H]-iloprost, 10 pL assay buffer or iloprost, and 200 & membranes. Adding the components in this order minimizes the risk of

cross-contamination(seeNote 11).

Keen

4

4. Following the addmon of the membranes, mix each sample well, and leave at room temperature for sufficient time for equilibrium to be achieved, m this case, 30 min (see Note 12). Reserve leftover membranes for protein determination, and dilutions of radioligand for countmg (see step 8, below) 5. If using an unfamiliar radiohgand, it is advisable to perform filter blanks, m which the total and nonspecific bmdmg of the radiollgand is determined m the absence of any membranes (see Note 13). 6. At the end of the incubatton period, separate bound llgand from free ligand In this case, filter the samples onto GF/B glass fiber filters using a Brandel cell harvester and rapidly (see Note 14) rinse the filters with 3 x 3.5 mL cold buffer (see Note 15). 7. Place the filters m 5-mL scmtillatton vials (see Note 16) and add 4 5 mL emulsifying scmtrilant. Leave the vials overnight to allow the radiohgand to be extracted from the filters into the scintillant (see Note 17) 8 Prepare radioligand standards. Pipet 10 pL of each radroligand dilution mto 5-mL scmtillatron vials and add 4 5 mL scmttllant. These provide an accurate estimate of the actual amount of radtoligand added to each sample. 9 Count the samples and standards m a scmtillation counter. 10 This basic method can be easily modified to examine the effects of modulators on radiohgand bmding Thus, to examme the effects of 100 p&Y GppNHp on [3H]-tloprost binding, as here, mitially add only 20 pL assay buffer to each tube and then add 10 p.L GppNHp The rest of the assay components are then added as described in step 3, above

3.4. Competition (Displacement) Binding In a competttion-bindmg assay,the bmding of an unlabeled ligand is measured by its ability to displace the specific binding of a low, fixed concentration of radioligand. This is an extremely useful and versatile techmque, allowing the properties of a wide range of ligands to be investigated rapidly, even those with rather low affinities for the receptor; it 1swidely used as a primary screening technique in drug-discovery programs. Furthermore, as the selectivity of the technique is determined by the selecttvtty of the radiohgand, it allows the study of the binding of nonselective hgands to one particular receptor of interest. 1. The design of the experiment again depends on the chotce of separation method. In the case of a 24-place cell harvester, as used here, it is convenient to set up each binding curve as follows: 4 nM [3H]-iloprost alone, to determine total binding (see Note 18); 4 nA4 [3H]-iloprost plus 10 concentrations of unlabeled ligand (see Note 19), m the case of the experiments presented here, 0.1-3 pA4 iloprost;

and4 nA4[3H]-iloprost plus 10pMunlabeled iloprost to define nonspecific bindmg (each determmation being performed in duplicate) 2 Dilute the ligands and perform the assay exactly as descrtbed for dtrect bmdmg (see Section 3 3.) Remember to count 10 pL of the radioligand dilution as a concentration standard and retain excess membranes for protein determmation.

Radioligand-Binding

Methods

5

3.5. Data Analysis A potential danger of radioligand binding is that the very simplicity of the technique may obscure the need for careful experimental design and thoughtful analysis of the data. It IS easy to obtain reproducible data, feed it into a computer, and get values out. It is much harder to be sure that those values really mean what you hope they mean. As with all data analyses, the principle of rubbish in, rubbish out applies; if the data are highly scattered or riddled with artifacts, any estimates of receptor number or affimty generated by even the most sophisttcated of curve-fittmg techniques will be flawed. A detailed consideration of the avoidance of potential artifacts is beyond the scope of this chapter, but is considered in some detail elsewhere (1,3). However, even if the data that go into the analysis are perfectly good, it is still possible to get rubbish out. The hazards of using linearizations of bmdmg data, such as the Scatchard plot, are widely recognized (4,5), and these techniques should be avoided. However, the use of nonlinear, curve-fitting techniques also has problems; as the number of variable parameters in the various models of binding increases, the number of combinations of these values that will fit the data more or less equally well also increases greatly. There is no guarantee that the purely mathematical, nonlinear regression analysis will automatically converge on the biologically correct combination. It is thus helpful to consider this sort of analysis as testing a hypothesis (se> Note 20) rather than automatically yielding precise and meaningful values. For effective curve fitting, it is necessary to start with simple models, only increasing their complexity if the data demand; keep the underlying assumptions of the various models m mind, checking that the analysis gives sensible results; and check the results of the analysis for consistency with results from other experiments. In order to illustrate these principles, the following sections consider in some detail the processes involved m analyzing some real data-the binding of [3H]-iloprost to platelets and NGlO8-15 cells. In the case of [3H]-iloprost, analysis of competition-binding curves is somewhat more straightforward than direct binding, so these will be dealt with first. 3.5.1. Analysis of Competition-Binding Curves I. Calculation of radioligand concentration. The precise amount of tracer radioligand included in eachsampleis determinedfrom the lo-& standard,as follows: radiohgand (pmol/sample)= (standarddpm)/(specific activity x 2220) where the specific activity of the radioligand is expressedasCMnmol and 2220 is a conversion factor This amountcan beconverted to a concentrationby taking the samplevolume into account.In this case,10 pL radioligand was included in

Keen

F

100 -

F .ma IE" .a 8 e

50 -

s P ..-P c c z

ooI

I

I

1

1 o-l0

1o-g

IO8

lo-'

[iloprost]

(M)

I

I

IO9

1o-6

Fig. K The inhibition of the specific binding of 7.3 nA4 [3H]-iloprost by unlabeled rloprost m the absence (A) and presence (8) of 100 pi14 GppNHp in human platelet membranes. The solid line represents the best fit of a single-site model of binding to the data obtained in the absence of GppNHp; the dotted line represents the best fit of the same model to the data obtained in the presence of GppNHp (see Section 3.5.1. for details).

a final assay volume of 250 &. Thus the radioligand concentration in nM(=pmol/mL) is given by: [radioligand]

(nM) = (standard dpm x 4)/(specific activity x 2220)

2. Calculation of % inhibition of specific binding. The data presented here (Figs. 1 and 2) are expressed as % inhibition of specific binding of [3H]-iloprost, which IS calculated as follows: % inhibition

= 100 x { I- [(sample dpm - nsb dpm)/(total dpm - nsb dpm)])

where sample dpm is the mean dpm in each sample containing a particular concentration of the unlabeled lrgand, total dpm is the mean dpm in the samples containing [3H]-rloprost alone, and nsb dpm is the mean dpm in the samples containing 10 p.M unlabeled rloprost to define nonspecific binding. This transformation of the data IS useful in that it incorporates total and non-

Radioligand-Binding

Methods

7

specific binding into the inhibition curve so that low concentrations of unlabeled ligand should inhibit 0% specific binding and high concentrations inhibit 100%. However, the transformation is by no means essential and may incorporate errors, if estimates of total and nonspecific binding are not accurate (see Note 2 1). 3. The effect of GppNHp on iloprost binding to platelet membranes. a. The inhibition of the specific bindmg of 7.3 nA4 [3H]-iloprost by unlabeled iloprost in the absence and presence of 100 pM GppNHp is shown m Fig. 1. The first step in the analysis of any binding data should be to determine the best fit of the simplest model of binding to the data: the simple Langmuir isotherm, or single-site model. This model assumes binding to a single population of noninteracting sites, so that:

WI = &a, P-W + PI) where [LR] is the concentration of ligand-receptor complexes (equivalent to % inhibition in this case), B,,, is the maximal binding capacity or total number of receptor sites (which should be 100% inhibition in the case of competition binding data), and K is an estimate of ligand-binding affinity (see part c, below). Curve fitting is performed by computer assisted, nonlinear regression analysis. A wide range of suitable programs are available (see Section 2.). Fitting the single-site model to the binding curves in Fig. 1 yielded the following estimates for K and B,,,: K B max

no GppNHp 2.00 x 10-8M 99.7%

+ GPPNHP 2.11 x l&s44 100.8%

b. Inspection of the predicted curves plotted in Fig. 1 shows that the curves tit the data rather well. Furthermore, the estimates of K and B,,, seem sensible; in each case B,,, is close to 100%, as expected, and the estimates of K obtained m the absence and presence of GppNHp are very similar, in accord with the observation that GppNHp appears to have very little effect on iloprost binding under these circumstances (see Fig 1). Thus the fit of the single-site binding model to the data seems satisfactory, and no further curve fitting IS required (or justified) in this case. c. Correction for the presence of [3H]-iloprost. Competition-binding curves, such as those shown in Fig. 1, are necessarily obtained in the presence of a low, fixed concentration of radiohgand. The presence of this radioligand affects the position of the binding curve for the unlabeled ligand; it is shifted to the right of its true position by a factor determined by the radiohgand aftinity and concentration. Thus, it is necessary to correct estimates of affinity obtained from competition studies to take this rightward shift into account, using the Cheng-Prusoff equation (6): true Kd = K/{ 1 + ([L*]/K*)}

IA 100

50

00 I

1O“O

I

I

1o-g

IO9

[iloprost]

I

IO.'

I

I

IO"

1o-5

(M)

IB A

I

I

I

1O"O

lo-g

1o-6

lo-'

[iloprost]

(M)

I

I

I

1O-e

1O'5

Fig. 2. The inhibition of the specific binding of 2.4 nM [3H]-iloprost by unlabeled iloprost in the absence (A) and presence (V) of 100 @4 GppNHp in NGl08-15 cell homogenates. The lines represent the best fit of various models to the data. (A) The dotted lines represent the best fit of a single-site model of binding; the solid lines represent the best fit of the same model, in which B,,, is constrained to 100%. (B) The solid lines represent the

IC .-g E 5 0 z.% 2

IOO-

50 -

.-s .tl P .5.$

O0 I

lo-I0

I

losg

I

IO-*

[iloprost]

1o-7

1

I

I

1o-5

lo-s

(M)

ID

;.-z 100 P 1

I

*

I

lo-lo

I

1o-g

A

I

I

I

I

IO4

1o-7

lo+

1o-5

[iloprost]

(M)

best fit of the Hill equation to the same data. (C) The solid lines represent the best fit of a two-site model of binding. (D) The solid lines represent the best fit of a single-site model of binding to the data obtained in the presence of GppNHp and the best fit of a two-site model of binding to the data obtained in the absence of GppNHp, in which K2 has been constrained to the value of K estimated in the presence of GppNHp (see Section 3.5.1. for details).

10

Keen where true Kd IS the corrected dissociation constant for the unlabeled ligand, [L*] is the radioligand concentration (determined from the radioligand standard; see step 1, above), andK* is the dissociation constant for the radioligand, determined from a direct-binding assay (see Section 3.5 2 ). In the case of a self-competition assay, as here, where the same ltgand is used as both radioligand and competing ligand, the Cheng-Prusoff equation simplifies to: true Kd = K - [L*] with the assumption that the affinities of the labeled and unlabeled form of the ligand are the same. Thus, for the iloprost binding to platelet membranes. Kd

no GppNHp 1.27 x 10-8M

+ G~PNHP 1.34 x 10-Q!

4. The effect of GppNHp on iloprost binding to NG108- 15 cell homogenates: a. The inhibition of the specific binding of 2.4 nM[3H]-iloprost by unlabeled iloprost in the absence and presence of 100 @fGppNHp is shown in Fig 2A. Again, the first step in the analysis of these data should be to determine the best fit of a smgle-site model to the data. This yielded the followmg estimates for K and B,,,, no GppNHp

K Bmax

6.06 x 10-gM 96.2%

+ GPPNHP 199x10-8M

95.2%

b. While the predicted curves fit the data points reasonably well, and the estimated K values reflect the fact that the curve obtained m the presence of GppNHp lies to the right of the curve obtained in its absence (see Fig. 2A, dotted lines), both estimates of B,,, are less than the expected value of 100% Thus, the sensible next step m the analysis is to see if these data are consistent with a B,,, value of lOO%, by fixing B,, to this value:

K Bmax

no GppNHp 6.90 x 10-9A4 100% (fixed)

+ GPPNHP 2.31 x lVsh4 100% (fixed)

The predicted curves given by this procedure are shown in Fig. 2A (solid lines). The curves still fit the data well and are thus likely to represent a better estimate of K than those obtained when B,,.,,,is allowed to converge on a value of less than 100% (see Note 22). c. The close agreement between the data and the single-site binding curves may suggest that any further analysis of this data using more complex models is unJustitied. However, the rightward shift in the binding curve obtained in the presence of GppNHp is characteristic of G protein-coupled receptors. It seems to reflect the ability of the guanme nucleottde to disrupt receptor-G protein complexes (RG), which have high affinity for agonists, converting them to uncoupled receptors (R), which have low agomst affinity. If the results of the

Radioligand-Binding

11

Methods

single-site fits are accepted at face value, they tend to suggest that, in the absence of GppNHp, 100% of the IP receptors exist as RG complexes and that all of these are uncoupled by GppNHp, to give a homogenous population of low-affinity agonist sites. While not impossible, this scenario is unlikely. Usually, in the absence of guanine nucleotides, agonists recognize a mixture of high- and low-affinity sites, reflecting a mixture of RG and R. In the presence of guanme nucleotide, the agonist-binding curve is shifted to the right and steepened, because of the conversion of high-affinity RG sites to lowaffinity R sites. From inspection of the data, it is obvious that GppNHp shifts the iloprost-binding curve to the right. The best way to determine the steepness of the curve is to fit the Hill equation to the data:

mJ4 = &l*d[L1”4L501”+ PI” where LSo is the midpoint location parameter of the curve (in this case the concentration of ligand inhibiting 50% of the specific binding) and n is the pseudo-Hill coefficient, or slope factor. If the data correspond to binding to a single population of nonmteracting sites, la is equal to 1 and the Hill equation simplifies to the single site model. If n > 1, it tends to indicate positive cooperativity between binding sites; if n < 1, it tends to indicate binding to a heterogenous population of sites (see Note 23). In many curve-fitting programs, the Hill equation is expressed in a slightly different format. For example, in FigP: amount bound = [min. + (max. - min.)]/{1 + ([L]/LSo)*) where min. is the minimum level of binding, obtained as ligand concentration tends to zero, and max. is the maximum level of binding, obtained as ligand concentration tends to 00. This form is particularly useful for fitting untransformed data and estimating levels of total and nonspecific binding (see Note 21). In analyzing the data from NG108-15 cells, min. and max. were constrained as 0 and loo%, respectively, and the best fit of the model to the data is shown in Fig. 2B:

L50

n

no GppNHp 6.88 x lo-9A!f 0.79

+ GPPNHP 2.30 x 10-8M 0.82

Thus, for both curves, the slope factor is less than one, indicating a degree of binding site heterogeneity. Furthermore, the curve obtained in the presence of GppNHp does appear to be slightly steeper in the presence of GppNHp than in its absence d. Based on the shallow slope factors and the working hypothesis regardmg the effects of GppNHp, the best fit of a two-site model to the data was determined. The two-site binding model is simply the sum of two single-site models:

WI = &ax1

[LY(& +

[LI) + 4nax2

M~2

+ [LI)

12

Keen The best fit of this model to the NG108-15 data is shown in Fig. 2C and yielded the followmg parameters: B maxl B max2 B maxl

+ Bmax2

4 K2

no GppNHp

+ GPPNHP

32.6% 65.5%

26.4% 75.2%

98.1% 135 x 10-9M 1.34 x 1tPM

6.85 x 10-9M 3.84 x 10-8M

101 6%

Inspection of Fig. 2C shows that the curves fit the data points well. Moreover, the sum of Bmaxl and Bmax2closely approximate the predicted value of 100%. However, these parameters do not support the hypothesis of conversion of RG sites to R by guanine nucleotides: The expected result would be the same values of K, and K2 m both the absence and presence of GppNHp, with only the relative proportions of the two sites differmg between the two curves Therefore, the next step in the analysts should be to determme how well the data can be described by a model that meets these criteria. e. There are curve-fitting packages available that allow the stmultaneous fitting of more than one curve, so that various parameters (e.g., KI and K2) can be shared between the various curves (see Note 24). However, the most widely available and user-friendly programs do not have this facility. It is, of course, possible to keep trying different combinattons of values for K1 and K2 until you hit on values that provide a good fit to both curves However, in this case, a somewhat simpler approach has been adopted. Given that the bindmg curve obtained in the presence of GppNHp can be well described by a single-site binding model, it is assumed that this curve represents binding to a predominantly homogenous population of low-affinity R sites. Thus the affinity value obtained from this single-site fit should at least approxtmate the affimty of the agonist for R (Kz m the two-site model) This approach gave the following values: no GppNHp B max 1 B max2 B maxl + Kl K2

&ax2

+ GPPNHP

54.6%

-

44.1%

100%

98.7% 2.42 x lW9M

-

2.31 x lo-s(fix)

2.31 x ltYsM

The predicted curves fit the data well (see Fig. 2D) and the sum of Bmaxl and B max2is close to 100%. The data can at least be said to be consistent with a

model in which guamne nucleotides promote the conversion of a subpopulation of high-affinity RG sites to low-affinity R sites. f. The final step is to convert the apparent affinity values from the competition binding experiments to true affinity values, as described above for the platelet data. Thus:

Radioligand-Binding

Kdl Kd2

13

Methods no GppNHp 0.02 x lo-sA4 2.07 x lO-%U

3.5.2. Analysis of Direct-Binding

+ GPPNHP 2.07 x lO+V

Curves

1. Calculation of radioligand concentrations These are determined as described in Section 3.5.1. 2 Calculation of specific binding. For each concentration of radtoligand, specttic binding 1s calculated as the difference between total and nonspecific binding. This obviously means that any errors in the determination of nonspecific binding will be incorporated mto the estimate of specific binding. In the vast majority of cases, nonspecific binding is a linear function of radtohgand concentration, and this can be used to check the accuracy of the nonspecific binding data. Many curve-fitting packages include a linear, “nonspecific binding” component in their one- and two-site models (see Note 25), and it is thus possible to estimate both specific and nonspecific binding from the total binding curve. However, this adds an extra level of complexity (and therefore variability) to the model and it is better to eliminate nonspecific binding at the beginning. The equation used to determine radiohgand concentration from dpm can then be used to convert the amount of radiohgand bound to pmol/sample. However, it is probably easier to leave these values as dpmsample until the end of the analysts, as here (Figs. 3 and 4) 3. The effect of GppNHp on the specitic binding of [3H]-rloprost to platelet membranes. a. The specrtic binding of 0.3-100 nA4 [3H]-iloprost in the absence and presence of 100 @f GppNHp is shown in Fig. 3A As previously, the first step in the analysis of these data is to determine the best fit of a single-site binding model (indicated by the solid lines in Fig. 3A):

Kd

B*ax

no GppNHp 19.0 nh4 4412 dpm

+ GPPNHP

27.6 ml4

4851 dpm

Note that in the case of direct binding experiments, the curve fits yield estimates of the true affinity of the radioligand; no correction of these values is necessary. Note also that, in the case of the direct-binding curves, GppNHp does appear to produce a small but consistent shift to the right of the agonistbinding curve, which was not apparent in the competition experiment (see Note 26). The predicted lines tit the data points well and the estimates of B,,, are reasonably similar, which fits in with the expected effects of GppNHp in this system. However, comparison of the Kd values with those obtained from the competition data (see Section 3.5.1.) suggest that the binding affinity of [3H]-iloprost is somewhat lower than that of unlabeled iloprost in the same membranes.

Keen

14 4000

3000

2000

1000

0 0.1

1

[“IH

10

dloprost

(nM)

4000

3000

2000

1000

0 1

[3H]-iloprost

10

(nM)

100

Radioligand-Binding Methods b. A logical next step in the analysis is therefore to see how well the directbmding data can be described by the model if Kd is constrained to the values obtained from the competition experiment (indicated by the dotted lines in Fig. 3A): Kd

B max

no GppNHp 12.7 nM (fixed) 3925 dpm

+ GPPNHP 13.4 nM (fixed) 3824 dpm

In this case the predicted lines do not fit the data particularly well, suggesting that the discrepancies between the Kd values in the competition and directbinding experiments cannot be simply caused by the vagaries of the curvefitting process. c. There is in fact a problem wtth using [3H]-iloprost m direct-binding curves, which underlies the apparent differences between affinity values obtained in direct and competition assays. At concentrations any higher than 10 nA4, a significant proportion of the iloprost-displaceable binding of [3H]-iloprost is to a low-affinity, high-capacity, nonreceptor site, which probably corresponds to the low-affinity prostanoid site found in many tissues (2,3). The presence of this second site ts more obvious m the direct-bmding curves obtained in NGl OS-15 cell homogenates (see point 4, below). It would be theoretically possible to account for this second site using a two-site model of binding. However, the two-site model has four variable parameters, and it is probably not justifiable to fit a data set of only 6 points (as here) to such a model. In practice, direct-binding curves for [3H]-iloprost can be well described by a single-site model, with the addition of a linear component (the component of nonspecific bmdmg included in this equation in many commercially available packages): [LR] = Bmax [L]/K,, + CL]/+ C[L] This tends to imply that, over the concentration range used, the occupancy of the second, nonreceptor site by [3H]-iloprost is very low. Under these conditions, the single-site binding model approximates to a straight line. The best fit of this model to the data is shown in Fig. 3B, the fits giving the following parameters:

Fig. 3. (previous page) The specific binding of [3H]-iloprost measured in the absence (A) and presence (V) of 100 pMGppNHp in human platelet membranes. The lines represent the best fit of various models to the data. (A) The solid lines represent the best fit of a single-site model of binding; the dotted lines represent the best fit of the same model, in which Kd has been constrained to the values obtained from the competition experiment. (B) The solid lines represent the best fit of a single-site binding model with the addition of a linear component to the same data (see Section 3.5.2. for details).

16

Keen 900

A

1

600

1

[3H]-iloprost

100

(rd~

900 -B

z B F E .IP

750 -

600 -

450 -

300 -

150 -

1

fH]-iloprost

(II;;

Radioligand-Binding Methods no GppNHp Kd

B max c

8.6

ml4

2641 dpm 15.6

17 + GPPNHP 27.6 nA4 2924 dpm 14.5

The predtcted curves tit the data very well, the two estimates ofB,,, are similar and the estimates of C (the nonreceptor component of binding) are very ahke m the absence and presence of GppNHp. This is as predicted, because guanine nucleotrdes would not be expected to affect nomeceptor bmdmg. Furthermore, the estimate of iloprost affhuty obtained m the absence of GppNHp corresponds closely to that obtained m the competition study (14.5 ml4 compared with 13.4 nMJ The correspondence between the estimates of affinity obtained in the absence of GppNHp are not as good, which is likely to reflect the fact that the binding curve obtained in the absence of guanine nucleotide is likely to represent bmdmg to a heterogenous population of RG and R sites, and the fact that the use of [3H]-iloprost as a tracer ligand in the competition study is likely to obscure this heterogeneity (see Note 26). It IS important to note that ignoring the existence of the nonreceptor sate, and takmg the single-site fits to the platelet data at face value, leads to a substantial overestimate of B,,, (the number of receptors in the tissue) and an underestimate of lloprost affinity. d. Knowing the specific activity of the [3H]-rloprost (14.7 Wmmol) and the concentration of protein in the homogenate used in the assay (0.8 mg/mL), the values obtained for B,,,= can now be converted from dpmlsample to finol.Jmg protein, as described m Section 3.5.1.: Bmax

no GppNHp 505 fmol/mg

+ GPPNHP 560 fmol/mg

e. If the purpose of these experiments was to investigate the effect of GppNHp on binding m more detail, it might seem appropriate to fit a two-site model of binding to the data, again incorporating a linear, nonreceptor component. However, this sort of analysis would require more experiments to be performed; there is an insufficient number of data points here to Justify the use of such a complex model. It would be necessary to obtain more detailed directbinding curves, with a greater number of data points incorporated over the same concentration range (see Note 19)

Fig. 4. (previous page) The specific binding of [3H]-iloprost measured in the absence (A) and presence (V) of 100 pM GppNHp in NGlOS-15 cell homogenates. The lines represent the best fit of various models to the data. (A) The solid lines represent the best fit of a single-site model of binding. (B) The dotted lines represent the best fit of a single-site binding model, with the addition of a linear component to the same data; the solid lines represent the best fit of the same model to the data, but, in the case of the data obtained in the presence of GppNHp, Kd has been constrained to the value obtained from the competition experiment (see Section 3.5.2 for details).

Keen

18

4. The effect of GppNHp on the specific binding of [3H]-iloprost to NG 108-l 5 cell homogenates. a. The specific binding of 0.3-100 nM [3H]-iloprost in the absence and presence of 100 @4 GppNHp is shown in Fig. 4A. The curve-fitting strategy used here is the same as that outlined above for the data from platelet membranes. Initially, a single-site model was fitted to the data: Kd

Bmm

no GppNHp 16.1 ti 925dpm

+ GPPNHP

79.8 ml4

1441 dpm

Several factors indicate that the use of the single-site model is not appropriate. The predicted curves do fit the data particularly well (see Fig. 4A), the estimates of affinity are substantially lower than those obtained from the competition study, and GppNHp appears to produce a 50% increase in receptor number. b. Fitting the data to a single-site model, with the addition of a linear component, gave predicted curves, shown as the dotted lines in Fig. 4B, and the following parameters: no GppNHp Kd

Bmax c

2.3 rut4

+ GPPNHP

2.7 nM

358 dpm

157 dpm

5.9

7.2

The predicted curves tit the data points much better than the simple singlesite model. However, it appears from this fit that GppNHp has very little effect on the affinity of [3H]-iloprost, but substantially reduces B,,,. While this effect of guanine nucleotide on the binding of a labeled agonist is possible (see Note 26), it is not what is expected in this case. Furthermore, the estimate of iloprost affinity obtained in the presence of GppNHp does not agree very well with that estimated in the competition experiment. c. In order to investigate whether these data are compatible with the competition data, the effect of constraining the affinity of [3H]-iloprost in the presence of GppNHp to the value obtained in the competition study on the fit of the singlesite model + linear component was examined: no GppNHp Kd

Bmax C

+ GPPNHP

2.3 ml4

20.7 nkf (fixed)

358 dpm

389 dpm

5.9

5.2

The predicted curves (solid lines in Fig. 4B) still fit the data well. Moreover, the estimates of B,,,,,are now very similar in the absence and presence of GppNHp, as is the value of the nonreceptor component of binding, C. This fit seems to represent the most acceptable compromise between a model that provides the best tit to the data and one that fits in with other information about the system. As before, the fit of more complex models to these data is not justified, because of the small number of data points.

Radioiigand-Binding

Methods

d. The protein concentration in homogenate used in this experiment 0.44 mg/mL; thus the following B,,, values can be calculated: B max

no GppNHp 125 fmol/mg

was

+ GPPNHP 135 fmol/mg

4. Notes 1. Further details regarding the growth of NGl OS-15 cells may be found in ref. 7. 2. Cold membrane suspensions tend to clump together, making accurate pipeting difficult. The suspension will be more homogenous if warmed to room temperature before pipeting. However, the membranes will deteriorate if kept at room temperature for too long. 3. If necessary, washed membrane preparations may be prepared from NGl OS-15 cells by several subsequent centrifugation and resuspension steps, as described for the preparation of platelet membranes (Section 3.1.). In this case, after the first centrifugation step, it is useful to filter the suspension through muslin, in order to remove the large tangle of DNA that will have formed. However, in the system used here, washed membrane preparations appear to offer no advantages over crude homogenates. 4. If required, NG108-15 cells can be harvested on the day of assay by agitation in phosphate buffered saline and suspended in an isotonic medium, such as gassed Krebs or cell culture medium, to allow the binding of ligands to intact cells to be measured. In the case of [3H]-.iloprost, this presents considerable problems: [3H]-iloprost is a very hydrophobic ligand that readily penetrates intact cells, thus leading to high levels of nonspecific binding. However, binding to intact cells can be extremely useful as a means of measuring binding under physiological conditions (81, or to investigate receptor internalization, using hydrophilic ligands that do not penetrate the cells (9). Nevertheless, the use of intact cells does represent an additional level of complexity compared with membrane preparations, and is more prone to artifacts; in particular, receptor-mediated internalization of ligands may give rise to a large component of apparently displaceable binding, and estimates of B,,, that increase with incubation time. 5. In order to define nonspecific binding, it is best to use an unlabeled ligand that is different from the radioligand, and ideally one that is as structurally distinct as possible. This minimizes the risk of displaceable nonreceptor binding, to enzymes or uptake-sites, for example. However, this is not always possible, either because no suitable ligands exist, or because their use would be prohibitively expensive, especially since they need to be used at concentrations sufficient to occupy all the available receptors, even when competing with the highest concentration of radioligand. 6. Choice of separation method. A wide variety of possible separation methods are available, including equilibrium dialysis, gel filtration, centrifugation, filtration, and so on. The choice depends on the receptor preparation (e.g., gel filtration may be most suitable for soluble or solubilized receptors; simple rinsing may be best for receptor autoradiography), as well as the radioligand used. For example,

20

Keen

if the ligand has a rather low affinity for the receptor, significant amounts may drssoctate durmg filtratron; centrifugatton may be better, but is not appropriate if the ligand displays a htgh degree of nonspectfic bmdmg Of the various separation methods, filtration is the most wtdely used It is very fast, reproductble, and usually incorporates a washing step to reduce the amount of nonspecific bmdmg caused by loose association of the radtolrgand with membrane fragments, and so on. 7 The chotce of radtohgand may be a rather trivial matter; there may only be one available. In the event of a choice, high-affinity bgands are generally preferred; they can be used at lower concentrations, which tends to reduce both the cost and the level of nonspecific binding, and they are less likely to dissociate from the receptor durmg the separation procedure. However, if the affinity of the radiohgand 1stoo high (Kd C 0.1 nM), it may take an impractically long time for equilibrium to be achieved. Furthermore, the level of nonspecific binding for different ligands in different tissues may vary enormously; the only way to find the most appropriate ligand may be by trial and error. Antagonist hgands are generally preferred to agonists, partly because antagomsts often exhibit higher affinity than agonists, and partly because the binding of agonists tends to be more sensitive to the assay conditions. For example, the bmdmg of an agonist to a G protein-coupled receptor will depend on the state of receptor G protein couplmg, and the binding of an antagonist will not. It is obviously best if the radrohgand displays a high degree of selecttvtty for the receptor of interest However, if necessary, it may be possible to suppress bmdmg to unwanted receptors by mcluding a saturating concentration of an unlabeled ligand that 1shighly selective for that receptor. [3H]-iloprost does not bind exclusively to the IP prostanoid receptor; it also exhibits high affinity for the EP, receptor (20). The more selective IP receptor ligand, cicaprost, can be used to determine whether this EP, receptor bmdmg presents a problem in any particular tissue; tf cicaprost inhibits significantly less than 100% of the specrfic bmdmg of [3H]-iloprost, it is hkely that the remamder represents binding to the EP, receptor. Fortunately, EPi receptor binding does not appear to be a problem m either platelets or NG108-15 cells. 8 The choice of radtoligand concentrations m a saturation experiment depends partly on the separatton method to be used (which dictates the number of samples that can be conveniently incorporated mto a single-bmdmg curve) and partly on the question to be addressed. In this case, the different concentrations of [3H]-iloprost have been chosen to be separated by half-decades on a log scale and span the expected affinity of [3H]-tloprost, approx 10 nM If the affinity of the radiobgand is unknown, rt may be necessary to perform range-finding experiments, with fewer concentrattons over a wider range. In order to tit complex binding models to the data, it would be necessary to include a greater number of ligand concentrations over the concentration range of interest. 9 Choice of assay buffer Radiohgand binding can usually be performed m very simple buffer systems. The choice depends on the preparation (e.g., whole cells require isotonic oxygenated medmm), the bgand (e.g., it may be necessary to perform binding in a high ionic strength buffer to reduce nonspecific

Radioligand-Binding

11

12.

13.

14.

15.

16.

17.

21

to filters; Mg2+ ions are essential for the binding of [3H]-iloprost and the question to be addressed (e.g., Mg2+ ions are required for the interaction between receptors and G proteins). The precise conditrons of the bmding assay (temperature, ionic composition of buffers, and so on) can affect ligand affinity; it is important to consider this when comparing data from different laboratories It is possible to lose substantial amounts of some particularly sticky ligands (both labeled and unlabeled) that may adhere to the sides of the tubes durmg dilution and during the assay. It may be possible to reduce this by using silamzed glass tubes, or by including bovine serum albumin in the buffer for peptide ligands, and so on. One of the largest sources of error in radioligand-binding experiments is poor pipetmg technique. It is important to add low concentrattons of ligands before high ones, to ensure that all additions reach the bottom of the assay tube, and to mix everything thoroughly. Sufficient time needs to be allowed for equtlibrium to be achieved, but this needs to be balanced against the stability of the receptor preparation and/or the ligands. Receptor stability can be improved at low temperatures, but this will also slow the rate of approach to equilibrium. Eqmlibrmm is approached most slowly at the lowest concentrations of radioligand and m competition assays. A fuller discussion of the problems of nonequtlibrmrn binding may be found in ref. 12. Radioligands commonly bind to filters; this contributes to measured level of nonspecific binding. It is often possible to reduce the level of filter bmdmg by, for example, increasing the ionic strength of the assay buffer or presoaking the filters in 1% polyethylenetmine. Filter binding presents a particular problem if it is displaceable by the unlabeled ligand used to define nonspecific binding, m this case it may be mdistmguishable from receptor binding. The rapid removal of free ligand during filtration necessarily disrupts the equilibrium and promotes dissociation of ligand from the receptor This loss can be considerable with low-affinity ligands, rendering filtration unsuitable. In all cases, the impact of dissociation should be minimized by filtering and rinsing the membranes as rapidly and reproducibly as possible, usmg icecold buffers throughout. Ideally, the filters should be rinsed with the assay buffer. However, the procedure gets through a great deal of buffer, and buffered tap water (cold tap water with a dash of buffer, to bring its pH to 7.4) usually works Just as well. The filters should be put close to the bottom of the vial to ensure that they are covered by scintillation fluid-but not scnmched up, because this slows the rate at which the radioactivity can be extracted by the scintillant. Following absorption of the scintillant, the filter becomes transparent and does not appear to interfere with counting. However, make sure that any inhomogeneity filter on the scintillation counter is switched off, otherwise, all the samples may be reJected. binding

[Zl]),

10.

Methods

22 18. The concentration of radioligand in competition studies needs to be kept as low as possible, to minimize the shift of the competition curves (see Section 3.5. l.), while still obtaining sufficient binding to allow the degree of inhibition of this binding to be accurately determined. The concentration of receptors in the assay may also need to be considered. When a low concentration of a high-affinity ligand binds to an abundant population of receptors, the free-ligand concentration may he substantially reduced, a phenomenon known as depletion. If the freeradioligand concentration is reduced by more than 10% by depletton, the receptor preparation needs to be diluted. 19. An advantage of the competition technique is that the unlabeled ligands used can be of low affinity or rather nonselective. The choice of concentration range depends on the same factors as considered above for radioligands in a directbinding assay (see Note 8). 20. Testing a hypothesis is exactly what curve fitting is, in that It involves determming the best fit of various theoretical models to the data. The choice of model is therefore, all important, and it is worth bearing in mind that the underlying assumptions of the model may or may not be completely accurate. For example, the two-site model used here to analyze agonist-bindmg assumes the existence of two populations of noninterconverting sites. However, RG can clearly be converted to R under appropriate conditions, such as the addition of guanine nucleotides. Curve-fitting techniques that do not rely on an underlying model have been described (13). 2 1. Calculation of % inhibition of specific binding assumes that the estimates of total and nonspecific binding are absolutely accurate; but, of course, they may not be. It is possible to obtain independent estimates of total and nonspecific binding by determining the best fit of the Hill equation to the untransformed data, in which case total and nonspecific binding will correspond to min. and max. respectively (see Section 3.5.2.). 22. If an unlabeled ligand consistently inhibits less (or more) than 100% of the specific binding of the radioligand, it may suggest that something is wrong with the definition of nonspecific binding. Inhibition of less than 100% of specific binding may legitimately occur in the case of an unlabeled ligand, highly specific for a particular receptor subtype, inhibiting binding of a nonselective radioligand to a heterogeneous population of receptors. It may also occur if the unlabeled ligand inhibits radioligand binding by an allosteric mechanism, in which both ligands can bind simultaneously to the receptor (14). 23. Steep curves (n > 1) can indicate that the ligand exhibits positive cooperatlvity. However, such curves can also arise artifactually, either because binding did not reach equilibrium (12) or because of ligand depletion (15). Shallow curves (n < 1) usually indicate binding-site heterogeneity, but can also arise from negative cooperativity. 24. A generally applicable program that enables the simultaneous fitting of several curves is available as part of BMDP (BMDP Statistical SoRware, Cork, Ireland). However, the use of this package is rather cumbersome and requires a working knowledge of Fortran.

25. It is important to constrain this linear, nonspecific binding component to zero in most cases (for example, when nonspecific binding has already been subtracted from the data, or in competition-binding studies, in which the nonspecific binding of the unlabeled ligand is necessarily invisible). 26. Binding-site heterogeneity will be obscured in competition studies in which the radioligand used exhibits selectivity for one of the populations of sites. For example, as an agonist, [3H]-iloprost has higher affinity for RG than for R. Thus, the rightward shift of the high-affinity component of the competition curve for an unlabeled agonist will be greater than for the low-affinity component. Hence, the competition curve is steepened. In extreme cases, low concentrations of labeled agonists will only bind to high-affinity RG sites. In this case, guanine nucleotides appear to produce a decrease in the B,,, for the labeled agonist, with no change in agonist affinity.

References 1. Hulme, E. C., ed. (1992) Receptor-Ligand Znteractzons. A Practical Approach. IRL Press at Oxford University Press, Oxford, UK. 2. Keen, M. and MacDermot, J. (1993) Analysis of receptors by radioligand binding, in Receptor Autoradiography: Principles and Practice (Wharton, J. and Polak, J. M., eds.), Oxford University Press, Oxford, UK, pp. 23-56. 3. Keen, M. (1995) The problems and pitfalls of radioligand binding, in Methods in Molecular Bzology, vol. 41. Szgnal Transduction Protocols (Kendall, D. A. and Hill, S. J., eds.), Humana, Totowa, NJ, pp. 1-16. 4. Klotz, I. M. (1982) Numbers of receptor sites from Scatchard graphs: facts and fantasies. Science 217, 1247-1249. 5. Burgisser, E. (1984) Radioligand-receptor binding studies: What’s wrong with the Scatchard analysis? Trends Pharmacol. Sci. 5, 142-144. 6. Cheng, Y. C. and Prusoff, W. H. (1973) Relationship between the inhibition constant Kl and the concentration of inhibitor which causes 50% inhibition (I&e) of an enzymic reaction. Biochem. Pharmacol. 22,3099-3 108. 7. Kelly, E., Keen, M., Nobbs, P., and MacDermot, J. (1990) Segregation of discrete G,,-mediated responses that accompany homologous or heterologous desensitization in two related somatic hybrids. Br J. Pharmacol. 99,309-3 16. 8. Nathanson, N. M. (1983) Binding of agonists and antagonists to muscarimc acetylcholine receptors on intact cultured heart cells. J. Neurochem. 41, 1545-1549. 9. Sibley, D. R. and Lefkowitz, R. J. (1985) Molecular mechanisms of receptor desensitization using the P-adrenergic receptor-coupled adenylate cyclase system as a model. Nature 317, 124-129. 10. Wise, H. and Jones, R. L. (1996) Focus on prostacyclin and its novel mimetics. Trends Pharmacol. Sci. 17, 17-21. 11. MacDermot, J., Blair, I., and Cresp, T. M. (198 1) Prostacyclin receptors of a neuronal hybrid cell line; divalent cations and ligand-receptor coupling. Biochem. Pharmacol.

30,2041-2044.

24

Keen

12. Motulsky, H. J. and Mahan, L. C. (1984) The kinetics of competitive radioligand binding predicted by the law of mass action. Mol. Pharmacol. 25, l-9. 13. Tobler, H. J. and Engel, G. (1983) Affinity spectra: a novel way for the evaluation of equilibrium binding experiments. Naunyn-Schmiedeberg’s Arch. Pharmacol. 322,183-192.

14. Stockton, J. M., Birdsall, N. J. M., Burgen, A. S. V., and Hulme, E. C. (1983) Modification of the binding properties of muscarinic acetylcholine receptors by gallamine. Mol Pharmacol. 23,55 l-557. 15. Wells, J. W., Birdsall, N. J. M., Burgen, A. S. V., and Hulme, E. C. (1980) Competitive binding studies with multiple sites: effects arising from depletion of free radioligand. Biochim. Btophys Acta 632,464-469.

Site-Directed Mutagenesis and Chimeric Receptors in the Study of Receptor-Ligand Binding Mark E. Olah and Gary L. Stiles

’

1. Introduction Prior to the cloning of G protein-coupled receptors (GPCRs), structure-function analysis of the ligand-binding properties of these receptors had for the most part been limited to study of the structure-activity relationships (SAR) of agonists and antagonists at the individual receptors. For a particular receptor, these SAR data were obtained via synthesis of series of chemically modified hgands, followed by the determination of their binding activities in tissues or cells that natively expressed the receptor of interest. With the molecular cloning of a multitude of GPCRs over the last several years, the amino acid sequence of these receptors is now known. With this knowledge, structural features of the receptors that are involved in ligand binding may now be explored in mutagenesis studies. Indeed, a principal focus of receptor research over recent years has been the detailed structure-function analysis of the ligand-binding properties of genetically engineered receptors heterologously expressed in the appropriate cell systems. Through such studtes, receptor regions and even single amino acids involved in ligand recognition have been identified. This chapter details the techniques used in our laboratory for the construction and analysis of receptors possessing single ammo acid point mutations and receptors composed of amino acid sequence derived from two parent wild-type receptors, i.e., chimeric receptors. Specifically, we have used these techniques to study the structural features responsible for the binding properties of different adenosine receptor subtypes (1-3). Similar approaches have been used by many laboratories focusing on many different GPCRs (reviewed in refs. 4-6). From

Methods m Mo/ecu/ar Slology, vol 83 Receptor SIgnal Transduction Edlted by R A J Chalks Humana Press Inc , Totowa, NJ

25

Protocols

26

Oiah and Stiles

1.1. Design of Mutated Receptors 7.1.1. Point Mutations The rationale for selection of single amino acids for targeting in mutagenesis studies of ligand binding may be based on several factors. If the receptor is a member (species homolog or distinct subtype) of a receptor family in which other subtypes have been analyzed in mutagenesis studies, it may be of initial interest to determine if any conserved amino acids have a similar function in that particular receptor. However, if studies are being initiated on a receptor of interest on which there is no information from previously performed mutagenesis studies, targets may need to be selected based on less direct data. The chemical structure of ligands recognized by the receptor may suggest the nature of amino acids to be targeted. For example, adrenergic receptors were the first GPCRs to be examined in mutagenesis studies. Because catecholamines possessing a nitrogen that can be protonated are the prototypical agonist ligands for this receptor family, it was reasoned that a negatively charged amino acid of the receptor may serve as a counter ion for this critical functional group (4). It was subsequently shown that substitution of asparagine for an aspartate residue in transmembrane domain 3 of the P-adrenergic receptor nearly abolished agonist binding (7). Amino acid targets may also be suggested by the sensitivity of receptor ligand binding to specific chemical treatments. For example, treatment of rat brain membranes with the histidine specific reagent diethylpyrocarbonate was demonstrated to perturb agonist and antagonist binding by the A, adenosine receptor (8). Site-directed mutagenesis of histidine residues in transmembrane domain 6 and transmembrane domain 7 of the A1 adenosine receptor has subsequently shown the rmportance of these residues in ligand binding (r). Computer models of the three-dimensional ligand binding pocket of GPCRs have been used to select potentially important amino acid residues that have been examined in receptor mutagenesis studies (9). A more indirect rationale for targeting an individual amino acid may include its conservation in a specific location in all members of a specific receptor family, thus suggesting a critical function. Once an amino acid has been targeted for site-directed mutagenesis, the replacement residue must be selected. Frequently, alanine is chosen as the residue with which to replace the wild-type amino acid. Alanine is of small mass, making it relatively less likely to substantially disrupt protein structure. Additionally, alanine is unhkely to form bonds with the ligand(s) under examination. However, it may be found that other residues are more suitable than alanine for the substitution. For example, replacement of a bulky amino acid, such as tyrosine, in a transmembrane domain with the smaller alanine may be found to disrupt overall receptor conformation. Thus, an amino acid of similar

size, but lacking the propensity to participate m similar chemical interactions with the ligand, may be a more appropriate selection. Frequently, it is of interest to make reciprocal point mutations in which individual amino acids are swapped among receptor subtypes or specieshomologs in an attempt to define the structural basis for distinct pharmacologic profiles of the wild-type receptors. Replacement of wild-type residues with additional amino acids is further discussed in Section 4. 7.7 2. Chimeric Receptors The vast majority of chimeric receptors employed to study ligand binding are constructed from two parent receptors that belong to the same receptor family. For example, the initial study employing chimeric proteins to map ligand-binding domains of GPCRs employed sequencesderived from the a2- and P2-adrenergtc receptors (10). In selecting the wild-type receptors that will constitute the sequence of the chimeric receptor, the degree of similarity in the wild-type receptors regarding pharmacological profiles and amino acid sequences must be considered. Ideally, the parent receptors may share nearly identical binding affinity for an individual compound or one class of ligands, but differ substantially in the affinity for a separate class of compounds. These distinctions may exist between different subtypes m a receptor family or perhaps between species homologs of the same receptor. For example, our laboratory has employed chimeric receptors composed of sequences derived from the bovine A, adenosme receptor and the rat A3 adenosine receptor to examine the structural requirements for ligand binding by adenosine receptors. The two wild-type receptors, as well as receptor chimeras derived from their sequence, bind the agonist ‘251-AB-MECA with very similar high affinity, thus permitting receptor quantification. However, the very different affinities of the parent adenosine receptors for certain other agonists, and all antagonists, has allowed for receptor domains recognizing these latter ligands to be identified through the study of chlmeric receptors (2,3). With both chimeric receptors and receptors possessing pomt mutations, the ability of constructs to maintain highaffinity binding of at least one radioligand is very advantageous. Such binding permits the analysis via competition-binding assaysof other classesof ligands that may display decreases in affinity of several orders of magnitude. Additional factors will influence which specific regions of the parent wildtype receptors to exchange in the construction of chimenc receptors. As with selection of the parent receptors, the specific receptors and ligands under examination must be considered when identifying the receptor segments to be swapped. For example, the binding of most small ligands to their receptors, e.g , catecholamine binding by adrenergic receptors, appears to exclusively involve receptor transmembrane domains; receptor extracellular domains have

28

O/ah and Stiles

been shown to be involved in ligand recognition by several of the GPCRs that are activated by peptide agonists, such as the tachykinins (4,I1). Often, one of two systematic approaches is taken m the construction of chimeric receptors. First, a series of chimeras may be created with each construct focusing on a distinct transmembrane domain or extracellular region. Alternatively, an mltial study may examine one or two chimeric receptors composed of the replacement of multiple receptor regions, with the resulting data used to design subsequent chimeras of more defined substitution, Once the general receptor regions have been chosen for inclusion in a chimeric receptor, the precise splicing points should be selected. Of concern is the ability of the receptor to tolerate sequence substitution at these points. A major obstacle in the study of chimeric receptors is the lack of detectable expression of certain constructs, which obviously precludes analysis. Though rigid guidelines do not exist, it 1soften preferable to avoid initiating sequence replacements in receptor transmembrane domains. The probability of success with substitutions at precisely the interface of a transmembrane domain and an adjoining loop segment is perhaps slightly greater than that of the above option. In these situations, the chance of obtaining chimerit receptor expression may be increased if the sequences of the two parent wild-type receptors are highly homologous in the splicing region. Conversely, splicing in the loop regions of GPCRs is often well tolerated and may be the preferred strategy m the construction of initial chimeras. Additional factors in the design of receptors possessing point mutations and of chimeric receptors are discussed in Section 4. 2. Methods for Creation of Mutant Receptors The techniques employed in our laboratory for construction of receptors containmg point mutations as well as chimeric receptors utilize the polymerase chain reaction (PCR). For pomt mutations, the design of the oligonucleotide primers and strategy employed in the PCR reactions are based on the method for site-directed mutagenesis described by Nelson and Long (12). The construction of chimeric receptors follows a similar approach, but also mcorporates the use of splicing oligonucleotides as described by Yon and Fried (13). Thus, the methods for creating point mutations or chimeric receptors are relatively similar, with the construction of the latter requiring additional oligonucleotide primers and an additional PCR step. Described below in detail are the methods for construction of the two types of mutant receptors. The procedure for point mutations is described first because it is relatively simple; the construction of receptor chimeras is an extension of this procedure. Alternative techniques for the site-directed mutagenesis of GPCRs (14), as well as the creation of chimeric receptors (15), have recently been outlined in excellent reviews.

Receptor-LigandBinding

29

2.7. Point Mutations The template to be used in the construction of point mutations may be the isolated wild-type receptor cDNA of interest or, more frequently, the receptor cDNA subcloned into a vector such as pBluescript or an expression vector. In the latter instances, the construct does not need to be lineartzed prior to the PCR reaction. Prior to the design of the oligonucleotide primers, the receptor cDNA must be analyzed for the existence of two unique restriction endonuclease sites that flank the area selected for mutation. Following the PCR generation of the mutated fragment, these sites will ultimately be used for subclonmg the cDNA; thus, they should be absent from the expression vector to be employed. Preferably, sites as close as possible to the target region are selected because this limits the amount of DNA sequencing required to ensure that unwanted PCR errors have not been introduced during the PCR amplification steps. If two convenient unique restriction endonuclease sites are not present in the receptor cDNA, sites used for subcloning the construct into the vector may be employed. Infrequently, the limited number of useful unique restriction sites located in the receptor cDNA may also be present in the expression vector typically employed for transfection procedures, making subcloning via these sites inconvenient. In these situations, it may be necessary to employ a shuttle vector that lacks these restriction sites. The cDNA subcloned into the shuttle vector is used as the template in the PCR reactions. Following the PCR amplification, the mutated cDNA fragment is subcloned via the unique sites into the shuttle vector and then again subcloned into the expression vector of choice using additional convenient restriction sites. 2.1.1. Oligonucleotides Four oligonucleotide primers are required for the creation of point mutations, as will be described. In terms of expense, it is important to realize that three of the four oligonucleotides may be repeatedly employed to construct subsequent mutations, aslong as the receptor region of interest remains the same, i.e., the target sequence is flanked by the same two unique restriction sites selected initially. Additionally, these oligonucleotides are useful as primers in the sequencing reactions that follow subcloning and permit the relatively rapid determination of the fidelity of the PCR amplification. A schematic outline of the design of the oligonucleotides and the PCR reactions is provided in Fig. 1, The first oligonucleotide (primer A) will code for the desired point mutation and should be designed to permit annealing to the coding or sensestrand of the cDNA. Typically, the oligonucleotide conststs of approx 5-8 bases 5’ of the point mutation to be made and -15-20 bases3’ to the region of interest. Figure 2 is an example of the design of an oligonucleotide specifying the mutation of a

O/ah and Stiles

30 ENDONUCLEASE SITE 1

ENDONUCLEASE SITE 2

5’

3’

PCR REACTION 1

5 -11

-b

FRAGMENT 1

3

PCR REACTION 2A --

+

PCR REACTION 28 3

5

FRAGMENT 2

ENDONUCLEASE SITE 1

ENDDNUCLEASE SITE 2

Fig. 1. Schematic representation of the construction of a receptor possessing a point mutation as described in the text. Letters (A-D) in circles represent the required four oligonucleotide primers. The diamond in primer A represents the base(s) coding for the mutation. Stippled region of primer B is that of dummy sequence. Arrows indicate direction of PCR amplification. Not all intermediate DNA fragments are depicted. Triangles indicate unique restriction endonuclease sites to be used in subcloning the final PCR product.

histidine residue to alanine. Design of this oligonucleotide and the others employed in this procedure should permit a theoretical melting temperature (T,,,) of at least 50°C, thereby lessening the occurrence of primers annealing to additional segments of the cDNA template and reducing the amplification of unwanted cDNA segments. Oligonucleotides consisting of 18-20 bases with

Receptor-Ligand

WILD-TYPE

5' 3'

37

Binding RECEPTOR

SEQUENCE

- AGC TGG CTG CCT TTG CAC ATC CT - 3' - TCG ACC GAC GGA AAC GTG TAG GA - 5' SER TRY LEU PRO LEU HIS ILE

To replace histidine (CAC) with alanine (GCC):

5'3'-

AGC TGG CTG CCT TTG a ATC CT - 3' TCG ACC GAC GGA AAC CGG ATC CT - 5'

The oligonucleotide

5'-

TC CTA u

selected for the PCR reaction is:

CAA AGG CAG CCA GCT - 3'

Fig. 2. Example of the design of an oligonucleotide coding for a point mutation (HISTIDINE TO ALANINE). Sequencespecifying the mutation IS underlined. -60% G/C content should provide for this minimum T,. The use of ohgonucleotides with segments of self-complementary sequence should be avoided, The second oligonucleotide (primer B) consists of approx 36 bases. The 18 bases composing the 3’ end of this oligonucleotide should be complementary to 18 bases of sequence of the antisense strand of the cDNA template upstream of restriction endonuclease site 1. A distance of W-100 bases between the oligonucleotide target sequence and restriction endonuclease site 1 is typically employed. The 18 bases composing the 5’ end of primer B are of sequence unrelated to that of the DNA template. This dummy sequence should be of -60% G/C content and again have a T,.,,of at least 50°C. Primer C is an oligonucleotide consisting solely of the 18 basesof dummy sequence contained in primer B. Primer D, designed to contain at least 18 bases, should anneal to the sense strand of the DNA template approx 5&100 bases downstream of restriction endonuclease site 2. 2.1.2. PCR Reactions For the creation of point mutations in the cDNA, 2 PCR reactions are required. PCR reaction 1 consists of -100 ng cDNA template (typically in 10 &L); 200 pM each of dGTP, dATP, dTTP, and dCTP (dNTP mix); 50 pmol primer A; 50 pmol primer B; polymerase buffer; thermostable DNA polymerase; and Hz0 to a final volume of 100 pL. Standard commercially available thermostable DNA polymerases may be employed. Our laboratory has employed both Vent DNA Polymerase (New England Biolabs [Beverly, MA]) and Tuq DNA Polymerase (Life Techologies, Gaithersbwg, MD) with equiva-

32

Olah and Stiles

lent results. The amount of the polymerase, the buffer concentratton, and any other additional components, e.g., MgC12, should be employed as directed by the manufacturer. The PCR parameters typically employed for PCR reaction 1 are 95°C x I mm; 50°C x 1 min; 72OC x 1.5 min. The program is run for 25 cycles and IS termtnated with a single 72°C extension conducted for 10 min. In almost all instances, we have found 25 cycles of amplification to provide sufficient quantities of product. Approximately 20 & of the reaction mix is electrophoresed on an agarose gel (l-l .5% depending on the size of the expected band) and the DNA fragment of interest (fragment 1) purified by standard techniques. Our laboratory employs elution of the DNA fragment mto a well that has been cut mto the agarose gel directly below the band of interest. For the elution procedure, the well contains O.lMammonmm acetate. Other rsolation methods, such as the commercially available Qiaex (Qiagen [Santa Clorita, CA]), also work well. The isolated DNA is extracted once with isoamyl alcohol, ammonium acetate/ethanol-precipitated, and resuspended in 50 pL H20. This DNA fragment is then used as a primer in a single-cycle PCR extension step as follows: PCR reaction 2A: -100 ng cDNA template (typically in 10 JJL); 10 pL of purttied DNA fragment 1; 100 @4 dNTP mix; polymerase buffer; thermostable DNA polymerase; and HZ0 to final volume of 50 &. The PCR parameters are 95°C x 2 min; 45°C x 2 min; and 72°C x 10 min. This program is run for a single cycle during which fragment 1 is extended with the receptor cDNA used as template. Upon completion of the IO-min extension, the thermal cycler should be programmed to initiate a cycle consisting of the same parameters as those used in PCR reaction 1. During the first 95’C denaturation step, the additional components for the reaction (PCR reaction 2B) should be added. The components are 50 pmol primer C; 50 pmol primer D; 100 w dNTP mix; DNA polymerase buffer; thermostable DNA polymerase; and HZ0 to a final volume of 50 &. Typically, these components are prepared during the lo-min extension of PCR reaction 2A and then introduced in a single 50-a addrtion directly under the mineral oil of the PCR reaction tube, which remains in the thermal cycler. PCR reaction 2B is continued for 25 cycles, with a final 72’C extension step conducted for 10 min. The product of these two PCR reactions is a DNA fragment (fragment 2) containing the desired point mutation, flanked by two unique restriction endonuclease sites, which permit subclonmg mto an expression vector. The inclusion of the dummy sequence in primers B and C ensures that only the DNA containing the desired mutation is amplified, and not wild-type receptor cDNA. The location of the dummy sequence upstream of restriction endonuclease site 1 permits its removal during subcloning. Approximately 10 pL of the product of PCR reaction 2B may be electrophoresed on an agarose gel to

Receptor-Ligand Binding

33

determine if a fragment of the expected size has been amplified and to check for the presence of any additional bands. The remainder of the product should be extracted with phenokchloroform, ammonium acetate:ethanol-precipitated, and digested with the appropriate restriction endonucleases. The fragment may then be ligated into the appropriate vector using standard molecular biological techniques. E. coli are transformed and plated, colonies grown up in media, and mimprep DNA obtained via standard methods (16). The presence of the desired mutation is determined by DNA sequencing. All DNA derived from PCR amplification, i.e., that spanning restriction endonuclease sites 1 and 2 should be sequenced to ensure that unwanted mutations have not been introduced durmg the procedures. Plasmid DNA may then be isolated on a large scale, e.g., Plasmid Maxi Kit (Qiagen), for transfection into cells of interest. 2.2. Chimeric Receptors As mentioned previously, creation of receptor chimeras via the PCR approach is similar to that described (Section 2.1.) for point mutations, with the requirement of an additional oligonucleotide and an additional PCR step. To illustrate the design of oligonucleotides and the PCR steps, an example will be employed m which the third and fourth transmembrane domains (and connecting sequence) of receptor A (acceptor) will be replaced with the analogous regions of receptor D (donor). A schematic outline of the design of the oligonucleotides and PCR reactions is provided m Fig. 3. Initially, it is often helpful to obtain a computer-generated alignment of the amino acid sequences of receptor A and receptor D for the determination of splicing junctions. 2.2.1. Oligonucleo tides As described for the creation of point mutations, the cDNA of receptor A must first be analyzed for the existence of two unique endonuclease sites that flank the region of interest, i.e., transmembrane domains 3-4. Again, these sites will ultimately be employed for subclonmg the engineered DNA fragment into receptor A. Regarding restriction endonuclease sites and receptor D, it is only a concern that the two sites selected for subcloning do not exist in the sequence of receptor D that is to be used in the substitution. Two oligonucleotide primers are required to amplify the region of receptor D selected for the substitution into a precise region of receptor A. These two primers thus define the splicing junctions. The first primer (primer A) consists of 36 bases, with 18 bases of the 3’ end designed to anneal to the antisense strand of receptor D. These 18 bases should code for the initial sequence of receptor D that will be contained in the chimeric receptor, i.e., the first six amino acids of the third transmembrane domain. The 18 bases at the 5’ end of primer A should be complementary to the antisense strand of receptor A in the region after which

Olah and Stiles

s Pm REACTION

3

3

5

3 4----

2A

s

3

5

T

5’

3

FRAGMENT 3 3

FRAGMENT2

3 5 3

A

A5

5 T 5

Fig. 3. Schematic representation of the construction of a chimeric receptor as describedin the text. Openbar representsDNA sequenceof donor receptor, Solid bar is DNA sequenceof acceptorreceptor. Letters (A-E) in circles representthe required five oligonucleotide primers. Stippled region of primer C is that of dummy sequence. Arrows indicate direction of PCR amplification. Not all intermediateDNA fragments are depicted. Triangles indicate unique restriction endonucleasesitesto be used in subcloning the final PCR product.

sequence substitution will be initiated, i.e., the last six amino acids of the first extracellular loop. Primer B consists of 36 bases,with the 18 basesof the 3’ end complementary to the sense strand of receptor D in the region at which substitution will be terminated, i.e., the last six amino acids of transmembrane domain 4. The 18 bases composing the 5’ end of primer B should be complementary to the sense strand of receptor A in the region at which receptor A sequence will be reinitiated within the chimeric receptor, i.e., the first six amino acids of the second extracellular loop. For both primer A and primer B, the 18 bases originating from receptor A and receptor D sequence should always provide for a T, of at least 50°C. If required to obtain this minimum T,,,, the number of bases may be increased. The remaining three oligonucleotide primers required for construction of the chimeric receptor are analogous to those employed in the creation of receptor point mutations. Primer C is composed at the 3’ end of 18 bases complementary to the antisense strand of receptor A upstream of restriction endonuclease site 1 and at the 5’ end of 18 bases of dummy sequence. Primer D is composed solely of the 18 bases of dummy sequence. Primer E is complementary to the sense strand of receptor A downstream of restriction endonuclease site 2.

Recepfor-Ligand Sinding

35

2.2.2. PCR Reactions The first step in constructing the chimeric receptor is the amplification of the sequence of receptor D that will compose the substitution (PCR reaction 1): -100 ng receptor D cDNA; 200 pM dNTP mix; 50 pm01 primer A; 50 pmol primer B; DNA polymerase buffer; thermostable DNA polymerase; and Hz0 to 100 pL final volume. The PCR parameters are the same as those employed in PCR reaction 1 in the creation of point mutations. After the 25 cycles of PCR, approx 20 @ of the product may be electrophoresed on an agarose gel and the fragment of interest (fragment 1) purified. The DNA is extracted once with isoamyl alcohol, ammonium acetate:ethanol-precipitated, and resuspended in 50 pL of H20. Fragment 1 is composed primarily of the sequence of receptor D, with sequence of receptor A at both ends, which should permit annealing to the receptor A cDNA template in the succeeding steps. Fragment 1 is then used as a primer in the second PCR reaction (PCR reactton 2A): -100 ng receptor A cDNA; 10 & fragment 1; 100 pM dNTP mix; DNA polymerase buffer; thermostable DNA polymerase; and Hz0 to a final volume of 50 pL. The PCR parameters are 95°C x 2 min; 45OC x 2 min; and 72°C x 10 mm. This program is run for one cycle only. Upon completion of the 10 min extension, the thermal cycler should be programmed to initiate a cycle consisting of the same parameters used in PCR step 1. During the initial 95OCdenaturation, additional components are added (PCR reaction 2B): 50 pmol primer B; 50 pmol primer C; 100 @4 dNTP mix; DNA polymerase buffer; thermostable DNA polymerase; and Hz0 to a final volume of 50 pL. As in the constructron of the receptors possessing point mutations, these components are introduced in a single 50-pL addition directly to the PCR reaction tube beneath the mineral oil. PCR reaction 2B is run for 25 cycles, with a final lo-min extension. Again, an aliquot of the reaction mix is run on an agarose gel and the fragment of interest (fragment 2) is purified, extracted with isoamyl alcohol, precipitated, and resuspended in 50 pL, of H20. Fragment 2 is then used as a primer in the initial part of PCR reaction 3A: - 100 ng receptor A cDNA; 10 pL fragment 2; 100 @I dNTP mix; DNA polymerase buffer; thermostable DNA polymerase; and Hz0 to a final volume of 50 pL. The PCR parameters are identical to those used in PCR reaction 2A. Upon completion of the extension step, additional components are then added to the reaction tube in a final volume of 50 pL (PCR reaction 3B): 50 pmol primer D; 50 pmol primer E; 100 @! dNTP mix; DNA polymerase buffer; thermostable DNA polymerase; and H,O to a final volume of 50 &

36

O/ah and Stiles

The PCR parameters are the same as those used for PCR reaction 2B (25 cycles). Ten microliters of the product may be run on an agarose gel to determine if a fragment of the expected size has been amplified and to ascertain the presence of any additional contaminating bands. The remainder of the reaction may be phenol:chloroform-extracted, precipitated, and digested with the appropriate restriction endonucleases for subcloning. The remaining procedures for preparation of the construct are exactly as described (Section 2.1.2.) for the creation of pomt mutations. For screening purposes, it is often convemerit and time-saving to perform a restriction endonuclease digestion of miniprep DNA to determine the presence of the chimeric receptor substitutton. Frequently, the substitution of DNA sequence results m the addition or removal of a restriction site, which is easily recognized relative to the wild-type receptor cDNA. Once identified, the isolated plasmid DNA may then be subjected to sequencing. 2.2.3. Additional Notes The following are factors that should be considered during the construction of chimeric receptors as outlined (Section 2.2.). Regarding primer B, it was noted that the sequence composmg the 3’ end of this ohgonucleotide should anneal to the sense strand of receptor D. The ability of this sequence to anneal to the analogous region of receptor A should also be determined. If this sequence of primer B is homologous to that of receptor A, we have observed m PCR reaction 2B the unwanted amplification of wild-type receptor A template rather than the desired amphfication of chimeric fragment 1 sequence. The greater the sequence homology displayed by receptor A and receptor D in the area near the splicing junction, the more likely this possibility becomes. Thus, if necessary in the design of primer B, the sequence from receptor D selected to compose the 3’ end of the oligonucleotide should be extended until the possibility of annealing to receptor A has been greatly reduced. Increasing the programmed annealing temperature m PCR reaction 2B may also be beneficial. We have used the method outlined (Section 2.2.) for the construction of chimeric receptors containing sequence substitution of as few as 20 amino acids. Accounting for the sequence of the acceptor receptor in primer A and primer B, this requires the amplificatton and puriticatton of an approx lOO-bp DNA fragment. For amplification of this limited amount of sequence m PCR reaction 1, we decrease the dNTP concentration to 100 @4 and the amount of primer A and primer B to 25 pmol. For the sequence substitutton of smaller receptor regions, e.g., 10 or fewer amino acids, we have successfully employed the protocol described previously for construction of point mutations. In these situations, a single oligonucleotide may be used to code for the multiple base changes. A single oligonucleotide may also be used to delete a limited amount

of nucleotide sequence if required. For this purpose, the primer should contain at least 15 bases complementary to the cDNA template on either side of the omitted sequence.

3. Expression of Mutated Receptor Constructs Following its construction, the binding properties of a mutated receptor, either a point mutation or chimeric receptor, may be studied by subcloning the cDNA into an expression vector and transfecting the construct into a suitable cell line. Many expression vectors are available and suitable for this procedure. In our laboratory, both pBC (17) and pCMV5 (18) have been employed. It is desirable to select an expression vector and cell line for transfection that permit for relatively high-level expression of the constructs. This is particularly beneficial for certain mutated receptors that may be much more poorly expressed than weld-type receptors. A system that permits only low levels of wildtype receptor expression may not be useful for analysis of mutated receptors that are present at several-fold lower levels. Perhaps the most frequently used system for the study of the ligand-binding properties of mutated receptors are COS cells that have been transiently transfected with the receptor cDNA-expression vector construct. COS cells permit for the replication of DNA vectors that contain the SV40 origin of DNA replication (16). In our laboratory, COS-7 cells are transiently transfected via a DEAE-dextran transfection procedure. The DEAE-dextran transfection procedure is that commonly used (161, with minor modifications. All solutions should be filter-sterilized prior to use. Day 1: COS-7 cells at approx 90% confluence in a T75 flask are washed one time with 10 mL of PBS and 5 mL of a PBS solution containing both 2.5 mg DEAE-dextran and the appropriate amount of DNA (see nextpage) is added to the flask. The flask is returned to the 37°C cell culture incubator for 30 min. At this time, 20 mL of DMEM/lO% FBS containing 100 @4chloroquine is added to the flask, which is then incubated at 37°C for 2.5 h. At this time, the media is removed and replaced with 5 mL of 10% DMSO in DMEM/lO% FBS. After an incubation of 2.5 min at room temperature, the media is removed and a fresh 25 mL of DMEM/I 0% FBS is added. Day 2: Media is removed and a fresh 25 mL of DMEM/lO% FBS is again added. The cells may be prepared for ligand-binding analysis on either d 3 or 4. Advantages of employing the DEAE-dextran transient transfection protocol include the relatively short amount of time required between obtaining the mutated receptor construct and determination of its ligand-binding properties. Additionally, through titration of the amount of receptor DNA-expression vector construct used in the protocol, it is possible to regulate to some degree the level of receptor expression. For example, transfection of cells with 5-l 0 pg of

38

O/ah and Stiles

wild-type receptor DNA may result in membrane expression of 1 pmol/mg receptor protein. A similar amount of a mutated receptor that is expressed less efficiently may perhaps be obtained if the amount of receptor cDNA used in the transfection is increased to 25 or 30 g. Since with certain mutated receptors there may be a plateau at which no increased receptor expression is obtained, even with increasing amounts of transfected cDNA, it may be necessary to titrate down the level of expression of wild-type receptors or of those mutants that may express more efficiently than wild-type receptor. Such a regulation of receptor levels may be particularly desirable when studying agonist ligand binding in which the amount of expressed receptor, and hence the stoichiometry of receptor to G proteins in the cell or membrane preparation, may theoretically influence agonist affinity. Generally, the quantities of expressed receptor obtained with transiently transfected COS-7 cells are greater than those present in native tissues and are more than adequate for receptor-ligand bmding studies via saturation binding analysis or competition bmding assays. Radioligand-binding analysis of mutated receptors heterologously expressed in mammalian cells may be performed via standard methods. Conditions for the assay, such as the use of intact cells vs membrane preparations, incubation temperature, and time, are typically predetermined using the wild-type receptor and are dependent on factors such as ligands available, cell type employed, and factors intrinsic to the receptor under analysis. In certain instances (see Section 4.), analysis of the functional or biochemical activity of a mutated receptor may be required to assessits ligand-binding properties. For example, the potency of an agonist ligand in eliciting a biochemical response may be used as an indicator of agonist affinity. In these situations, the cell type to be used, as well as the transfection procedure (i.e., transient vs stable transfection), is governed by the known signaling properties of the receptor being examined. For example, transient transfection of mutated receptors in COS cells may be used for receptors that couple to phospholipase C activation via the G, class of G proteins; stable transfection of receptor constructsmay be required to demonstratethe receptor-mediated inhibition of adenylyl cyclaseoccurring via activation of a member of the G, family. Stable expression of a receptor may also be desired for functional studiesbecausethis permits a source of cells in which the receptor population IS constant over time. In transient transfections, the expression of receptors may vary from day to day, with disparate expression among cells obtained from a single transfection. 4. Analysis and Interpretation of Results This chapter primarily focuses on the techniques used in receptor mutagenesis. It may, however, be appropriate to conclude with a discussion of factors pertaining to the analysis of data obtained from ligand-binding studies with

Receptor-ligand Binding

39

genetically engineered receptors. The goal of these studies is to identify receptor regions or perhaps specific amino acids that are involved in Iigand recognition by the receptor. When a mutation is found to alter ligand binding relative to the wild-type receptor, the most pertinent question is whether the effect is direct or indirect. The observed response may occur as the result of a substitution of an amino acid(s) that directly binds the ligand via a chemical interaction such as hydrophobic or hydrogen bonding. Alternatively, the targeted amino acid may not actually interact with the ligand, but rather the substitution may indirectly affect ligand recognition by altering receptor conformation or architecture. At present, these possibilities may not be differentiated with absolute certainty. No single experiment permits the unequivocal assignment of the role of a particular receptor region or amino acid in ligand binding. However, the design of the initial mutagenesis experiments and the completion of complementary studies may be very valuable in obtaining the most valid insights from the data generated in the mutational analysis of ligand binding. In the study of receptors possessing point mutations and chimeric receptors, an initial consideration is the specificity of the effect of the sequence substitution. For example, disruption of agonist binding while antagonist binding is unaltered is an indication that the mutation may be specifically affecting an agonist-binding domain and is not perturbing overall receptor conformation. Observation of the loss of both agonist and antagonist radioligand binding is more difficult to interpret. It is not unreasonable to assume that certain amino acids of a receptor may be involved in the coordination of both classes of ligands. Thus, a single mutation may perhaps directly perturb the binding of both classes of ligands. However, it is also possible that the mutation disrupts receptor processing or folding and is preventing membrane insertion of the receptor. In such a situation, it may be possible to examine the activity of an agonist in a functional assay that permits the use of much higher ligand concentrations than those employed in radioligand-bmdmg assays. Additionally, many investigators have used immunologic techniques to demonstrate membrane expression of constructs that do not display ligand binding. Presumably, the alteration in the ligand-binding pocket produced by the mutation does not prevent recognition by the appropriate antibody. Demonstration of cell surface expression of a mutated receptor, however, does not preclude a disruption of receptor architecture that is nonspecifically altering ligand binding. In the design of genetically engineered receptors, particularly chimeric receptors, it is often advantageous to demonstrate a gain of function as well as a loss of ligand binding upon sequence substitution. For example, when studying receptor subtypes with substantially different binding profiles, it may be

40

Olah and Stiles

possible that a limited amount of sequence replacement in a chimeric receptor may significantly enhance its affinity for a particular class of ligands relative to the parent wild-type receptor from which the majority of its structure is derived. Relative to a loss of binding, such an enhancement may less likely result from an indirect effect on receptor structure. In this regard, the demonstration of reciprocal effects with reciprocal sequence replacements is an additional indication of direct effects of the mutation. In the study of receptors possessingpoint mutations, additional information regarding the precise role of a specific amino acid may at times be obtained if a series of amino acids is used to perform the substitution. As noted previously, in initial mutagenesis studies designed to determine if a particular amino acid is involved in ligand binding, the target residue is frequently replaced with alanine. The rationale for this selection is that the physical properties of alanine make it less likely than other residues to nonspecifically disrupt protein structure (particularly in transmembrane domains), and this residue has little propensity for forming chemical bonds with the ligand. A loss of binding upon alanine substitution is an initial indication of a role for the target residue in ligand binding. Additionally, many investigators have used the change in free energy of the binding interaction, i.e., the relationship of d(dG) = -RT ln(K,[mutant]/K,[wild-type], as an indication of the nature of the type of bond lost upon making a point mutation (19-21). Subsequent experiments may attempt to determine if the presence of the wild-type residue may be mimicked by replacement with an amino acid possessing the ability to provide a similar interaction with the hgand. Examples of ammo acids that have been found to functionally substitute for one another to varying degrees in mutagenesis studies of hgand binding to GPCRs include asparagine-glutamine (22), serinethreonine (9,23), histidme-glutamine (24), arginine-lysine (25,261, and glutamate-aspartate (7). Obviously, such relationships may not be found in all receptors because several factors, such as differences in overall receptor architecture, slight differences in amino acid bulk, and variation in their propensity for bond formation, must be considered. The specificity of the mutation may be further explored by studying the binding of ligands in which key functional groups have been replaced or deleted at both the wild-type and mutated receptor. This analysis also may indicate with which functional group of the ligand the targeted amino acid interacts. Theoretically, a ligand in which a crucial functional group has been removed will demonstrate similar affinity at a wild-type receptor and a point mutant of this receptor in which the amino acid specifically interacting with the particular functional group has been replaced. At such mutants, there should be no further reduction in affinity for modified ligands relative to the wild-type receptor, since the binding interaction does not occur in either situation. This

Receptor-Ligand Bindmg

41

approach has been taken to identify specific amino acid-ligand functional group interactions at the PZ-adrenergic (27), M3 muscarinic (28), neurokinin-1 (20), and TRH (29) receptors. The approaches described in this section provide a means to interpret the results obtained from the study of ligand binding at mutated receptors. The findings from multiple studies such as these must be assimilated for valid hypotheses to be made. Additionally, the results from these experiments may be used to enhance computer-generated models of the ligand-binding pockets of GPCRs. Until high resolution of receptor structure by electron crystallography is obtained, mutational analysis provides the best means to study, at the most basic level, the structure-function relationshrps involved in ligand binding by GPCRs.

References 1. Olah, M. E., Ren, H , Ostrowskr, J., Jacobson,K. A., and Stiles, G. L. (1992) Clonmg, expression, and characterization of the unique bovine A, adenosme receptor. Studies on the Iigand binding site by sate-directed mutagenesis. J Bzol Chem 267, 10,764-10,770. 2. Olah, M. E , Jacobson, K. A., and Stiles, G. L (1994) Identification of an adenosme receptor domain specifically mvolved m binding of 5’-substitutedadenosine agonists.J Bzol Chem 269, 18,01&18,020. 3. Olah, M. E., Jacobson, K. A , and Stiles, G L. (1994) Role of the second extracellular loop of adenosme receptors in agonist and antagonist binding. J. Bzol. Chem 269,24,692-24,698. 4. Fong, T. M. and Strader, C. D. (1994) Functional mapping of the ligand binding sites of G-protein coupled receptors. Med. Res Rev 14,387-399.

5. Baldwin, J. M. (1994) Structureand function of receptorscoupled to G proteins. Curr. Opzn. Ceil Bzol 6, 180-190. 6. Jacobson, K. A. and van Rhee, M. A. (1996) Molecular architecture of G proteincoupled receptors. Drug Dev Res. 37, l-38. 7. Strader, C. D., Sigal, I S , Candelore, M. R., Rands, E., Hill, W. S., and Dixon, R. A. F. (1988) Conserved aspartic acid resrdues 79 and 113 of the /3adrenergm receptor have different roles in receptor function. J. Bzol. Chem 263, 10,267-10,271. 8. Klotz, K.-N., Lohse, M. J., and Schwabe, U. (1988) Chemical modification of Ai adenosinereceptorsin rat brain membranes.J Biol. Chem 263, 17,522-17,526. 9. Kim, J., Wess, J., van Rhee, A. M., Schoneberg, T., and Jacobson, K. A. (1995)

Site-directedmutagenesisidentities residuesinvolved in ligand recogmtion in the human Aaa adenosine receptor. J Bzol. Chem 270, 13,987-13,997. 10. Kobilka, B. K., Kobilka, T. S., Damel, K., Regan, J. W., Caron, M. G., and Lefkowitz, R. J. (1988) Chimeric 02-, b2-adrenergic receptors: delmeation of

domainsmvolved m effector coupling andligand binding specificity.Sczence240, 1310-1316.

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11. Gether, U., Johansen, T. E., Snider, R. M., Lowe, J. A., Emonds-Alt, X., Yokota, Y ,, Nakanishi, S., and Schwartz, T. W (1993) Binding epitopes for peptide and non-peptide ligands on the NKl (substance P) receptor. Regal. Peptides 46,49-58. 12. Nelson, R. M. and Long, G. L. (1989) A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal. Biochem. 180, 147-15 1. 13. Yon, J and Fried, M. (1989) Precise gene fusion by PCR. Nucleic Acids Res. 17,4895. 14. Fong, T. M., Candelore, M. R., and Strader, C. D. (1995) Site-directed mutagenesis, in Methods in Neurosciences, vol. 25, Receptor Molecular Biology (Sealfon, S. C., ed.), Academic, San Diego, CA, pp. 263-277. 15. Suryanarayana, S. and Kobilka, B. K (1995) Receptor chimeras, in Methods zn Neurosczences, vol. 25, Receptor Molecular Biology (Sealfon, S. C., ed.), Academic, San Diego, CA, pp. 278-301. 16. Sambrook, J., Fntsch, E. F., and Mamatis, T. (1989)MoZecular Clonzng: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 17. Cullen, B. R. (1987) Use of eukaryotic expression technology in the functional analysis of cloned genes. Methods Enzymol. 152,684-704. 18. Andersson, S., Davis, D. L., Dahlback, H., Jornvall, H., and Russell, D. W. (1989) Clonmg, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J. Biol. Chem 264, 8222-8229.

19. Guan, X.-M , Peroutka, S. J., and Kobilka, B. K (1992) Identification of a smgle amino acid residue responsible for the binding of a class of j%adrenergic receptor antagonists to 5-hydroxytryptaminelA receptors. Mol. Pharmacol 41,695-698. 20. Fong, T. M., Cascieri, M. A., Yu, H., Bansal, A., Swain, C., and Strader, C. D. (1993) Amino-aromatic mteraction between histidine 197 of the neurokinin-1 receptor and CP 96345 Nature 362,350-353. 21. Perlman, J. H., Laakkonen, L., Osman, R., and Gershengorn, M. C. (1994) A model of the thyrotropin-releasing hormone (TRH) receptor bindmg pocket. J. Bzol. Chem. 269,23,383-23,386.

22. Chanda, P K., Minchin, M. C., Davis, A. R., Greenberg, L., Reilly, Y., McGregor, W. H., Bhat, R., Lubeck, M. D., Mizutani, S., and Hung, P. P. (1993) Identitication ofresidues important for ligand binding to the human 5-hydroxytryptamine,. serotonin receptor. Mol. Pharmacol. 43,5 16-520. 23. Townsend-Nicholson, A. and Schofield, P. R. (1994) A threonme residue in the seventh transmembrane domain of the human Al adenosme receptor mediates specific agonist binding. J. Bzol. Chem. 269,2373-2376. 24. Cascieri, M. A., Macleod, A. M., Underwood, D., Shiao, L.-L., Ber, E., Sadowski, S., Yu, H., Merchant, K. J., Swam, C. J , Strader, C. D., and Fong, T. M. (1994) Characterization of the interaction of N-acyl+tryptophan benzyl ester neurokinin antagonists with the human neurokinm- 1 receptor. J. Bzol. Chem 269,6587--659 1. 25. Perlman, J. H , Laakkonen, L., Osman, R , and Gershengorn, M. C. (1995) Distinct roles for arginmes in transmembrane hehces 6 and 7 of the thyrotropinreleasing hormone receptor. Mol. Pharmacol. 47,480484.

26. Zhou, W., Rodic, V., Kitanovic, S., Flanagan, C. A., Chi, L., Weinstein, H., Maayani, S., Millar, R. P., and Sealfon, S. C. (1995) A locus of the gonadotropinreleasing hormone receptor that differentiates agonist and antagonist binding sites. J. Biol. Chem. 270, 18,853~18,857. 27. Strader, C. D., Candelore, M. R., Hill, W. S., Sigal, I. S., and Dixon, R. A. F. (1989) Identification of two serine residues involved in agonist activation of the /3-adrenergic receptor. J Blol. Chem. 264, 13,572-13,578. 28. Wess, J., Maggio, R., Palmer, J. R., and Vogel, Z. (1992) Role of conserved threonine and tyrosine residues m acetylcholine binding and muscarinic receptor activation. J. Biol. Chem. 267, 19,3 13-19,3 19. 29. Perlman, J. H., Thaw, C. N., Laakkonen, L., Bowers, C. Y., Osman, R., and Gershengorn, M. C. (1994) Hydrogen bonding interaction of thyrotropin-releasing hormone (TRH) with transmembrane tyrosine 106 of the TRH receptor. J. Biol. Chem. 269,1610-1613.

3 Approaches to the Stable Transfection of G Protein-Coupled Receptors Andrea Townsend-Nicholson 1. Introduction 1.1. Why Use Stably Transfected Cell Lines to Study GPCRs? Heterologous expression in mammalian cell lines provides an invaluable tool with which to explore determinants of G protein-coupled receptor (GPCR) function. The use of stably transfected cell lines enables a receptor to be studied in the absence of other related receptor subtypes. With this technique, molecular determinants of ligand binding can be identified; signal transduction pathways of a particular receptor can be delineated; interactions between a transfected receptor and other expressed receptors, both endogenous and exogenous, can be established; interactions between a GPCR and different G proteins can be characterized; and biochemical modifications of GPCRs, such as phosphorylation, can be studied. Many different features of GPCR function can be explored through the use of stably transfected cell lines. One of the major advantages of stably transfected cell lmes is that a known, constant quantity of the transfected receptor is expressed for many generations. Both stably transfected pools and clonal cell lines can be studied. Stably transfected pools, or clonal cell lines, that expresshigh levels of a transfected receptor facilitate ligand binding studies; cell lines expressing physiologically relevant levels of a transfected receptor can be used to examine secondmessenger-couplmg pathways. In planning to establish a stably transfected cell line expressing a GPCR of interest, there are a number of different factors that must be taken into consideration. 1.2. Which Ce// Line to Transfect? The choice of cell line into which a GPCR is best transfected is very much dependent on the subsequent investigations to be performed. In general, it is From

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highly desirable that the cell line to be transfected has a reasonably rapid growth rate and a high transfection efficiency, and that it requires minimal maintenance. Because GPCRs are coupled to effector systems through heterotrimeric G proteins, it is important to ascertain that the appropriate G proteins and effecters are endogenously expressed in the cell line to be transfected. It is also helpful to determine if the cDNA to be transfected is already expressed endogenously in the cell line chosen for transfection. There are also other factors to be considered. Many GPCRs belong to families with more than one receptor subtype. If the ligands used for the study of these receptors are not suitably subtype-selective, it is possible for more than one receptor subtype to interact with a particular ligand. In this instance, it might be wise to choose a cell line devoid of endogenous receptors capable of interfering with the study of the transfected receptor. Sometimes, however, the choice of such a cell line is not always the best option. Our studtes of the four adenosine receptor subtypes, A,, AZa, AZb, and A3, have, for the most part, been carried out on stably transfected CHO.Kl cells (1-41, which do not appear to endogenously express any of the four known adenosine receptor subtypes. The human Aza adenosine receptor cDNA has been transfected into CHO.Kl cells a number of times and different stably transfected pools have been selected. In each of these, the A,, receptor binding was poor, as was the ability of the transfected receptor to stimulate the activity of adenylyl cyclase, the effector enzyme to which the AZareceptor is coupled. The same AZa receptor construct was used to transfect human embryonic kidney 293 cells and stably transfected pools were selected. The AZa receptor is expressed at high levels in 293 cells and the ligand binding and functional coupling profiles of this receptor are as would be expected (5). There appears to be something present in the 293 cells but missing in the CHO.Kl cells that is required for the correct function of the AZa receptor. Northern analysis of mRNA isolated from 293 cells reveals that the AZ,-,adenosine receptor subtype is endogenously expressed in these cells (Fig. 1). The AZb adenosine receptor subtype, like the AZasubtype, also increases CAMP levels through activation of adenylyl cyclase. 7.3. Which Expression Vector to Use? There are many good, commercially available vectors that will enable expression of cDNAs in mammalian cells. In selecting an appropriate expression vector, consideration should be given to the promoter that will be used for the expression of the cDNA of interest and to the selectable marker that will be used for the isolation of stably transfected cells expressing this cDNA. A promoter that will maximize expression in the cell line of choice should be chosen. Viral enhancer-promoter sequencesfrom the human cytomegalovi-

Stable Transfection of GPCRs

47

Fig. 1. Northern analysis of endogenous adenosine receptor expression in 293 cells, using probes derived from the coding region sequences of the Al, A2a, Azb, or A3 adenosine receptor subtype cDNAs. Two micrograms of poly A+ mRNA isolated from 293 cells were electrophoresed, transferred to a positively charged nylon membrane, and hybridized at high stringency with one of the four adenosine receptor subtype probes. The AZb receptor subtype probe hybridizes to a 1.6-kb transcript in 293 cells. No hybridization to the Al, Aza, or As receptor subtype probes is seen.

rus (CMV) immediate early gene,the simian virus (SV-40) early gene,andthe ROW sarcomavirus long terminal repeat(RSV LTR) are commonly used to achievea high level of constitutive expressionin a wide rangeof cell lines. It shouldbe confirmed that the promoterthat will be usedto expressthe receptor cDNA is active in the cell line that will be usedfor the transfection. There are several dominant selectablemarkers that provide a means for selectingstabletransfectants.Oneof the most common is neomycin (or G4 18) resistance(neoR),which is conferred by the product of the bacterial phosphotransferaseenzyme APH(3’)II. This marker renderscells resistant to the antibiotic G418. Another marker that can be used is hygromycin resistance, conferredby hygromycin-B-phosphotransferase, which renderscells resistant to the antibiotic hygromycin-B. Hygromycin resistanceis a useful marker for the transfectionof cells that are alreadyG418-resistant. 1.4. Transfection of Mammalian Cell Lines The majority of cells in a typical transfectionexperimentwill expressexogenousDNA transiently, and this transfectedDNA will be lost from the host cell after a number of cell divisions. In a small proportion of transfectedcells, the exogenousDNA will be integratedinto the chromosomalDNA of the recipient cell at oneor more randompoints. In theseinstances,the exogenousDNA hasbecomea stableelementof the genomeof the host cell and this cell is now stably transfected. There are two factors that affect the number of stable transfectantsthat can be obtained.The first is the efficiency with which the cells initially take up the exogenousDNA and the secondis the frequencyat which stableintegrationoccursoncethe exogenousDNA hasenteredthe cell.

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Because it is difficult to influence the latter, the number of cells that are stably transfected can be maximized by using conditions that result in a transfectton efficiency of the cells that is as high as possible. With the incorporation of a selectable marker in the exogenous DNA, it is possible to select for cells that have this DNA integrated mto their own chromosomal DNA. It should be noted that the expression of integrated exogenous DNAs is subject to the local environment at the site of integration. Strong expression can be obtained from even the weakest of promoters if integration occurs near a strong enhancer sequence. Similarly, expression from strong, constitutive promoters can be abrogated if integration occurs at the site of a transcriptional silencer. 1.5. Calcium Phosphate-Mediated of Mammalian Cells

Transfection

There are many different techniques by which exogenous DNA can be mcorporated into and expressed by mammalian cells. These include calcium phosphate-mediated and DEAE dextran-mediated transfection, electroporation, and the use of cationic lipophihc carriers. Calcium phosphate-mediated transfection is a very useful and simple technique for introducing significant quantities of DNA into a single cell. A large number of mammalian cell lines, particularly adherent cell lines, are able to be transfected with high efficiency using this method. Calcium phosphate-mediated transfection is also very useful for cotransfection studies, because more than one DNA molecule can be introduced into a single cell with reasonable efficiency. The calcium phosphate transfection protocol described by Chen and Okayama (6) gives highefficiency transfection of mammalian cell lines. 1.6. G418 Selection of Stably Transfected Mammalian Cells The amount of G418 needed to kill untransfected cells varies with different mammalian cell lines and should be determined for a given cell line in advance. When determined, this amount should then be used to select for stable transfectantsof this cell line. If greater amounts of G4 18 are used,then transfected cells expressing lower levels of the neo gene product may be killed, and selection will be biased toward cells expressing higher levels of the neo gene product. In our studies, wild-type and mutated adenosine receptor subtype cDNAs have been expressed under the control of the CMV promoter, using the expression vector pRc/CMV (Invitrogen), and transfected mto CHO.Kl cells, using the calcium phosphate method (6). Stably transfected pools have been selected using G418 resistance. Over 50 such pools have been established with this method. The range of receptor expression levels in these stably transfected pools varies from 14,000 to 480,000 receptor molecules/cell, although the upper and lower limits of this range were observed with mutated Al adenosine

Stable Transfecrion of GPCRs

49

receptors and may be caused, in whole or in part, by the particular mutation that was mtroduced.

2. Materials 2.1. Equipment 1. Standard tissue culture equipment, including a laminar flow hood and an inverted

microscope. 2. 5% COz incubator

3. 2--4% CO* incubator. 2.2. Disposable

Plasticware

1. Sterile pipets and other standard tissue culture materials. 2. Sterile 75- and 150-cm2 tissue culture flasks. 3 Sterrle test tubes (e.g., Falcon 2057 [Becton Dickinson, Bedford, MA]).

2.3. Reagents Note: Autoclaved MilhQ (Mlllipore) water should be used for the preparation of all solutions, 1. 2X BES-buffered solution (BBS): Prepare a stock of 2X BBS (50 mMBES [N,Nbls {2-hydroxyethyl} -2-aminoethanesulfonic acid] [Sigma, St. Louis, MO], 280 mM NaCl, 1.5 r& Na2HP04) and adjust the pH to approx 6 95 at room temperature using 1N NaOH. Filter-sterilize the solution using a 0.22-p filter (Nalge, Rochester, NY), ahquot, and freeze at -20°C. The aliquot currently in use is stored at room temperature after openmg. When first preparing this buffer, it is best to make 2X BBS at several different pH values (approx 6.7-7.2). These solutions should then be tested m transfection experiments to determme the optimal pH for transfection of the cell line in question. Subsequent preparations of 2X BBS should be set to this optimal pH value by using an ahquot of the original solution at the appropriate pH as a reference It is important to determine the pH of the 2X BBS solution at room temperature. 2. 2.5M CaCl,: Prepare a stock of 2.5M CaC12, filter-sterihze using a 0.22-p filter (Nalge), aliquot, and freeze at -20°C. The aliquot currently in use is stored at room temperature after opening. The use of tissue culture-grade calcium chloride for transfection solutions is not required. 3. G418 (Geneticin): Prepare a stock of G418 (Life Technologres, Galthersburg, MD) in sterile water, filter-sterilize using a 0.22-p filter (Nalge), aliquot, and freeze at -2O’C. Once opened, an ahquot may be kept for several weeks at 4°C. Stock concentrations of G418 are typically 100X, so if, for example, selection

with G418is to be carried out at 800 pg/mL, a stocksolution of 80mg/mL should be prepared. It is important to note that the amount of active G418 m each lot varies; the stock solution should be 80 mg/mL of active G4 18 and not 80 mg/mL of the mass of the G4 18 powder.

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4. Plasmid DNA: There are a number of different ways to prepare plasmid DNA, but CsCl-purified DNA always gives good results for both transient and stable transfection experiments. The purity of the DNA to be transfected is crucial. Traditionally, if the DNA was to be used for transfection studies, two CsCl-gradient purification steps were performed; however, this laborious procedure can be shortened by the use of a single CsCl-purification gradient followed by RNase A and Proteinase K digestion. After isolation of the plasmid band (it is important to take only the supercoiled DNA band), removal of the ethidium bromide, and dialysis to remove the CsCl, the DNA is digested with RNase A at a final concentration of 10 pg/mL for 60 min at 65°C. Proteinase K is added to a final concentration of 100 pg/mL and SDS to a final concentration of 0.2%, and the mixture is incubated for 60 min at 37°C. The DNA is then phenol-chloroform extracted, ethanol precipitated (using 250 mM NaCl), and resuspended in TE (10 mM TrisHCl pH 7.5, 1 mM EDTA) at a final concentration of 1 mg/mL. The ratio of the optical densities determined at 260 and 280 nm will give an idea of the purity of the DNA. Pure DNA has an OD,,dOD,s, ratio of 1.8; contamination with phenol or proteins will reduce this figure (7).

2.4. Cell Culture The CHO.Kl cell line is available from the American Type Culture Collection (Rockville, MD) and is successfully propagated in DMEM/Hams F12 (0.5X DMEM, 0.5X Hams F12, 10% fetal calf serum, 0.056% sodium bicarbonate, 2 mJ4 glutamine). CHO.Kl cells grow as an attached monolayer and can be detached using a solution of 1X PBS + 0.2% EDTA (10X PBS and 2% EDTA stock solutions were obtained from ICN Pharmaceuticals, Costa Mesa, CA). These cells are routinely passagedtwice weekly, seeding at 1:20 each time. 3. Methods The method described below is for the transfection and selection of CHO.Kl cells plated in 75-cm2 flasks (see Note 1). In these experiments, a mock-transfected control (which contains all of the components of the calcium phosphate DNA solution, with water in the place of the DNA) and an untransfected control should be included. 3.1. Preparation

of the Cells to Be Transfected

1. Establish a stock of exponentially growing CHO.Kl cells that have been passaged at least twice since being thawed. 2. Seed a 150-cm2 flask from this stock and grow to confluence. 3. From this confluent flask, plate cells to be transfected in 75-cm2 flasks at seeding densities of 1: 15 and 1:30 (typically, cells are detached from the surface of the 150-cm2 flask in 30 mL PBS/EDTA solution; either 0.5 mL [ 1:30] or 1 mL [ 1: 151 of this 30-mL mixture is added to 14 mL of medium in a 75-cm2 flask) (see Note 2). 4. Incubate cells at 37°C in 5% CO2 for approx 18-24 h.

Stable Transfection of GPCRs 3.2. Preparation

of the Calcium Phosphate-DNA

51 Solution

This solution can be prepared on the laboratory bench from sterile solutions; it is not necessary to prepare it in a laminar flow hood. The protocol below makes enough calcium phosphate-DNA solution for one 75-cm2 flask. 1. In a large sterile test tube, mix the following: 20 pg DNA, 50 pL 2.5M CaC12, sterile water to 500 pL. 2. Place the tube on a vortex-mixer with continual, gentle agitation. 3. Add 500 NIL 2X BBS, dropwise. 4. Mix thoroughly, but well. 5. Let mixture sit for l&20 min at room temperature.

3.3. Transfection of the Cells and Selection and Banking of the Stably Transfected Pool 1. Remove cells from incubator and add 1 mL of calcium phosphate-DNA solution to each 75-cm2 flask (the solution can be added directly to the medium in the bottom of the upright flask; it does not appear to be necessary to add the solution dropwise over the cells) (see Notes 3 and 4). Mix well with the medium in the flask. 2. Incubate cells at 37°C in 24% CO2 for 18-24 h (we routinely use 3% C02). 3. Remove cells from incubator; check appearance of precipitate and cells under inverted microscope (see Note 5). 4. Remove medium from flask and replace with 15 mL of fresh CHO.Kl medium containing G418 at a final concentration of 800 pg/mL. 5. Place cells at 37°C in 5% CO, until G41X selection is complete (see Notes 6-8). 6. Detach cells using PBS/EDTA solution, ensuring that there are no clumps. 7. Centrifuge gently for 5 min at 25OOg, resuspend in 30 mL CHO.Kl medium, and transfer to a 150-cm2 flask (see Note 9). 8. Incubate at 37°C in 5% CO2 for 24 h, or until cells are confluent. 9. Detach cells from flask and, following an appropriate protocol for preparing frozen stocks of cell lines, distribute in 3-5 aliquots/l50-cm2 flask and store in liquid nitrogen (see Note 10). At this stage, the stably transfected pool has been established and safely banked. One of the ampules can now be thawed for dilution cloning or for subsequent analyses.

4. Notes 1. Although a large number of transfections can be carried out simultaneously in Petri dishes (e.g., in 9-cm dishes), it is quite easy for a Petri dish lid to be knocked off accidentally in the incubator during the lengthy selection procedure. We have found it easiest to avoid problems of contamination during selection for stable transfectants by plating, transfecting, and selecting the cells in 75- or 150-cm2 tissue culture flasks.

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Townsend-Nicholson

2 It is important to determine the optimal density at which the cells should be plated in order to obtain reasonable transfectton efficiencies. The CHO.Kl cell line is most effectively transfected by the calcium phosphate method when the cells are plated to reach 40-60% confluence on the following day. The doubling time of CHO.Kl cells is approx 20 h. When the cells are plated too densely, they do not transfect well. When they are plated too sparsely, a heavy precipitate often forms and the cells will not survive. 3. Important areas to consider in optimizing transfection conditions include the pH of the 2X BBS solution, the amount and the purity of the supercooled DNA added to form the precipitate, the density of the cells at transfection, the pH of the media at the time of transfection, and the concentration of the CO2 to which the cells are exposed during the formation of the precipitate. If no colonies or few colonies are visible after selection is complete, then the transfection efficiency is too low and these variables will need to be manipulated to improve the efficiency of transfectlon. The possibtlity that the promoter used to express the receptor cDNA is macttve m the cell hne being transfected should also be considered. If many attempts at transfection are unsuccessful, tt may be that the cell line is refractory to transfection by the calcium phosphate method. 4. Many transfection protocols involve changing the medium several hours before putting the DNA-CaCl, precipitate on them. This step is both unnecessary and expensive because it increases the amount of medium to be used. If you wish to change the medium before transfection, however, the optimal pH of the 2X BBS solution will need to be determined under these conditions because it will be different from that required if the medium is not changed. 5. After the transfection, when looking at the cells under the microscope, there should not be a large quantity of precipitate visible. If the cells have large amounts of precipitate stuck to them, they will mevitably die, particularly if tt is vntually impossible to wash this precipitate off, A limited number of very tine grains seen in clear areas of the flask posttransfectron is a good indicator of a successful transfectton. Sometimes small, roundrsh, golden clumps are seen among the transfected cells, which is another good sign. 6. Some selection protocols include a step immediately after transfectron in which the cells are detached and replated before G418 treatment, at a density that will permit the isolation of single colonies after G418 selection. Spontaneous resistance to G418 can occur (the frequency of this can be calculated when the amount of G418 required to kill untransfected cells is being determined) and, if single colonies are to be isolated immediately, the frequency of spontaneous resistance to G418 should be taken into consideration when determimng the number of colonies to be chosen for the establishment of clonal lines. In order to maximize the number of stably transfected clones that can be isolated, it is best to select the many colonies contained in a stably transfected pool first and then to isolate clonal cell lines from this pool. 7. G418 is stable for 8-10 d at 37’C (personal communication, Life Technologies Techline), so the medium of selecting cells should be changed weekly and fresh

Stable Transfection of GPCRs

53

G418 added. More frequent changes are both expensive and unnecessary; less frequent changes will permit the growth of untransfected cells. When selecting stably transfected CHO.Kl cells, dead cells first start to appear m the medium approx 48-72 h after selection has commenced. 8. At the completion of G418 selection, numerous individual colonies will be visible in the flask containing the transfected cells. Although typical selection times for CHO.Kl cells range from 3 to 5 wk, selection can proceed more quickly and may be complete by 14-18 d. When a stably transfected pool has been selected this quickly, it can be banked and frozen if it is known that all of the untransfected cells will have died. By including and selecting either untransfected cells or mocktransfected cells with each transfection, it is possible to verify when all untransfected cells have died and, therefore, when it is safe to stop the G418 selection. 9. Cell lines do not need to be maintained in G4 18 once the stably transformed pool has been selected. If an established transfected line will be used for many passages, however, it is often useful to include G4 18 in the medium every now and then to verify that the cells are still G418-resistant. 10. Sometimes stably transfected cell lines lose transfected receptors. This may happen when the constitutive expression of a particular receptor IS deleterious to the growth of the cell. Difficulties such as this can be overcome by ensuring that a suitably sized frozen stock of the cell line is established at an early passage number and by thawing another ampule of this stock when needed. It may also be worth considermg the use of an expression vector that contains either an inducible promoter or specific steroid hormone response elements, which are able to confer steroid hormone-sensitive gene induction, so that the transfected receptor protein is expressed when required, instead of constitutively.

Acknowledgments I am grateful to Marianne Hallupp, Vikki Falls, Sharon Fielder, and the members of the Neurobiology Division of the Garvan Institute of Medical Research for their advice and assistance.

References 1. Townsend-Nicholson, A. and Shine, J. (1992) Molecular cloning and characterisation of a human brain Ai adenosine receptor cDNA. Mol Brain Res. 16, 365-370. 2. Pierce, K. D., Furlong, T. J., Selbie, L. A., and Shine, J. (1992) Molecular cloning and expression of an adenosine AZbreceptor from human brain. Biochem. Biophys. Res. Comm. 137,86-93. 3. Townsend-Nicholson, A. and Schofield, P. R. (1994) A threonine residue in the seventh transmembrane domain of the human Al adenosine’receptor mediates specific agonist binding. J. Biol. Chem 269,2373-2376. 4. Megson, A. C., Dickenson, J. M., Townsend-Nicholson, A., and Hill, S. J. (1995) Synergy between the inositol phosphate responses to transfected human adeno-

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and constitutive

P,-purinoceptors

in CHO-Kl

cells Br J

115,1415-1424.

5. Furlong, T. J., Pierce, K. D., Selbie, L. A., and Shine, J. (1992) Molecular characterization of a human brain adenosme A2 receptor. Mel Bram Res. 15,62--66 6. Chen, C. and Okayama, H. (1987) High-efficiency transformation of mammalian cells by ptasmid DNA. Mol. Cell Bzol. 7,2745-2752. 7. Sambrook J., Frttsch, E. F., and Maniatis, T. (1989) Appendtx in Molecular Clonzng* A Laboratory Manual, 2nd ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p, E5.

4 Methods for Transient Expression of Hetero-Oligomeric Ligand-Gated Ion Channels Hartmut Liiddens

and Esa R. Korpi

1. Introduction Transient transfectron of mammalian cell lines with recombinant DNA has become a common tool for studying functional and structural properties of a wide variety of proteins (I-5). Transient transfections are especially practical for the expression of recombinant multisubunit complexes becausetheir expression needs the prior knowledge and selection of which of a set of subunits are necessary to form functional heteromultrmers with distinct features. Subumt stoichiometry, mode of assembly,and the relative expression levels of individual cDNA vector constructs also have to be taken into account. The latter still poses an insurmountable problem, because no accurate predictions can be made. Several methods exist for transient and stable transfection of eukaryotrc cells. Calcmm phosphate precipitation (6) has gained wide acceptance.In recent years, other approaches have been developed. These include the gene transfer aided by lipopolycations (7-9), by electroporation (IO), and through bombardment with microprojectiles (11). They may be superior to the classical coprecipitation under certain conditions, but in our hands a modified version of the technique of Chen and Okayama (6) has proved to be efficient and reproducible for the generation of large amounts of the heteromultimeric y-aminobutyric acid type A (GABA*) receptor subtypes. The pharmacology and functional properties of GABA* receptors have been extensively studied in recombinant expression systems.Fifteen different subunits, grouped into the five classes--o(a l-6), p(p l-3), y(y l-3), 6, and p (p l-2)-have the potential to participate in the formation of a presumably pentameric GABA* receptor (12-1.5). A number of radiolabeled compounds can be used to investigate different regions and mnctronal aspectsof these receptors, e.g., From

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the benzodiazepine (BZ) site by [3H]-Ro 15-45 13, the GABA sites by [3H]muscimol, and the channel pore by [35S]-TBPS. Transfection of mammalian cell lines wrth one member of both the a and the l3 class of subunits is sufficient to yield high levels of functional GABA, receptors with [3H]-muscimol binding sites, but lacking a BZ binding site (‘26,17). BZ recognition sites can be formed only by the additional expression of a y-subunit (18). These ai l3j yk receptors (in which 1= 1-6, j = l-3, k = l-3) form Cl- channels activated by GABA and are allosterically modulated by BZ ligands (18). No receptors or channels are formed in the absence of a p variant (19,20). The following data illustrate that selective subunit concentrations affect the binding of recombinant GABA* receptors. Using equimolar amounts of expression vector for the three subunit classes a, p, and y, the resulting GABA*/BZ receptors can be detected by ligands to GABA and benzodiazepine sites. In addition, GABA-induced currents in these cells are modulated by BZ ligands (18,21,22). It is possible that coexpression of cd, pj, and y2/3 yields aipj receptors, in addition to aipjy2/3 receptors. Transfecting human embryonic kidney (HEK) 293 cells with equal amounts of expression vectors coding for the al- and P2-subunits, and increasing amounts of the y2 vector, lowered the level of [3H]-muscimol and [3H]-Ro 15-4513 binding (Fig. 1A), suggesting a complex interaction. With constant levels of p2 and y2 vectors, titration of the a6-subunit expression vector yielded bell-shaped curves for the binding of the three radioligands [3H]-muscimol, [3H]-Ro 15 4513, and [35S]-TBPS (Fig. 1B). In contrast, the binding of [3H]-muscimol, [3H]-Ro 15-45 13, and [35S]-TBPS increased with the amount of p2 expression vector added when titrated in the presence of constant levels of al and y2 vectors (Fig. 1C). Using an optimal vector ratio of 2: 10:0.2 for the ai, p2, and y2 expression vectors, the binding for [3H]-muscimol and [3H]-Ro 15-4513 increased to -3000 fmol/mg protein. [35S]-TBPS binding, measured at a tenth of the estimated K. value (23), increased from msignificant levels at high y2 concentrations to consistent levels of -50 fmol/mg protein at this vector ratio. These data exemplify the need for careful monitoring and optimization of the transfection protocol, with recommendable comparison to the features of native receptor populations (24,25). 2. Materials 2.1. For DIVA Maxi-Prep Procedure 1. Tris-EDTA buffer (100X TE): lMTris-HCI (60.57 g/500 mL), O.lMNa,-EDTA (16.81 g/500 I&), adjustpH to 7.5 with concentratedHCl. Sterilizeby autoclaving. Dilute aliquots with water to 1X TE.

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Fig. 1. Titration of a6, j32, and y2 expression vector in HEK 293 cells in lo-cm plates. Binding of 6 nA4 [3H]-muscimol (open bars, let? y-axis label), 6 nA4 [3H]-Ro 15-45 13 (vertical stripe, left y-axis label), and 6 nit4 [35S]-TBPS (horizontal stripes, right y-axis label) was measured. (A) HEK 293 cells were transfected with 5 pg each of the al and j32 expression vectors. The ~2 expression vector was added in the amounts stated. (B) HEK 293 cells were transfected with 0.2 ~18of r2 and 5 pg of p2 expression vectors. The a6 expression vector was added in the amounts stated. (C) HEK 293 cells were transfected with 0.2 pg of ~2 and 1 pg of al vectors. The g2 expression vector was added m the amounts stated.

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2. Terrific broth (TB): Prepare and sterilize by autoclaving: (a) 12 g bacto-tryptone (Difco, Detroit, MI), 24 g bacto-yeast extract (Difco), 4 mL glycerol made up to 900 mL by addition of distilled water; (b) 0.17A4KHZP0, (2.3 1 g/100 mL), 0.72M K,HPO, (12.54 g/100 mL). Add 100 mL of(b) to 900 mL of (a). Add the antibiottc needed just prior to use 3. Solution I (glucose): 50 mA4 Glucose (1% w/v) in 25 n&! Tris-HCl, pH 8.0, 1 mA4 EDTA (from OSMEDTA, pH 8.0, stock solution). 4 Solution II (NaOH/SDS) make fresh each time: Make up 1% sodium dodecylsulfate (SDS) (from a 10% SDS-stock solution) in 0.2MNaOH. 5. Solution III (potassium acetate solution): Mix 600 mL of 5Mpotassium, 115 mL glacial acetic acid, and 285 mL H,O. 6 CsCl-Solution: Dissolve 90 g CsCl in 90 mL 1X TE buffer 7. Saltwater-saturated tsopropanol: In a 500~mL screw-cap bottle, add NaCl to -100 mL H,O until saturated (an ample amount of salt should be visible) Then add 300 mL isopropanol and mix thoroughly. After -30 mm, three layers (salt, water, isopropanol) are clearly separated. (If salt plaques cover the bottle, add H,O until they resolve.) Fresh isopropanol can be repeatedly added. 8. OSM EDTA: Dissolve 84.06 g Na,-EDTA in -400 mL H,O. Adjust pH to 8.0 with lMNaOH, and adjust final vol to 500 mL by further addition of H,O. Sterilize by autoclaving.

2.2. For Transient Expression of 293 Cells on Petri Dishes 1. 293 MEM medium: To 500 mL MEM (Gibco, Gaithersburg, MD) add 1.l g NaHC4 (if medium IS not provided with NaHCOs), 50 mL heat-inactivated fetal calf serum (serum lots are mdividually checked from several suppliers; heat inactivate 50 mL of serum for 20 min at 56”(Z), 2 mMglutamme (commercially available as 100X stock [Gibco]), and 5 mL penicillin/streptomycin (commercially available as 100X stock solution). 2. 2X BBS (100 mL; keep 50-mL ahquots at -20°C solution m use at 4’C) 50 mM BES (N,N-bis[2-hydroxylethyll-2-ammoethanesulfonic acid; 1.07 g), 280 mM NaCl(1.63 g), 1.5 mA4NazHP04 * 2H,O (26.7 mg); add HZ0 to 100 mL, and stir for 15 min. Then carefully add 2M NaOH (-500 p.L) until the pH approaches 6.95 at room temperature (ensure that pH does not exceed 7.0; Important: Do not back-titrate: from pH < 7.0). The solution should be sterilized using a 0.2~pm filter apparatus. 3. 2MCaC12: 14.7 g CaClz * 2HzO per 50 mL HzO, store at 4’C. The solution should be sterilized using a 0.2~pm filter apparatus. 4. Mannitol buffer: 220 mMmannitol,70 mMsucrose, 4 WHEPES, 0.5 mMEDTA (from 0.5Mstock solution, pH 8.0). Adjust to pH 7.4 with NaOH and make up to 500 mL. Store at 4°C. 5. 10X PBS: 80 g NaCl, 2 g KCl, 14.4 g Na2HP04 * 2H,O, 2 g KH2P04 made up to 1 L; the pH should be 7.4 without further adjustment. Sterilize by autoclavmg. Dilute to 1X PBS prior to use with autoclaved Hz0

Note: All chemicals were at least analytical grade.

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3. Methods 3.1. DNA Maxi-Prep Procedure The large amount of DNA ueeded for some of the subunits requires the purification of the plasmids by the CsCl method. This yields large amounts of DNA in very high purity. We use column purification methods if only a small number of transfections are planned, or for pilot experiments. 1 Preculture 10-50 mL of transformed bacteria in TB-plus-antibtotic for -6 h. Add preculture to 200-250 mL TB-plus-antibiotic in stoppered 2-L baffled Erlenmeyer flasks, incubate overnight up to 24 h (see Note 1). 2. Centrifuge cultures (3OOOg, 10 min). Decant the supernatant and wipe remaining fluid off the walls. 3. Resuspend the pellet in 30 mL of Solution I by repeated tituration with a pipet. Incubate for 5 min at room temperature. 4. Transfer to a fresh 250-mL bucket and add 60 mL of Solution II. Mix cautiously by inverting the vessel several times (the mixture is very viscous) and incubate for 10 min on ice (see Note 2). 5. Add 50 mL of ice-cold Solution III and mix by inverting several times. Incubate for 5 min on ice. 6. Centrifuge (27,OOOg, 15 mm) and then filter supernatant through a Kimwipe in a funnel. Precipitate DNA with 100 mL tsopropanol (results in -250 mL solution) at room temperature and immediately centrifuge (27,OOOg, 20 min, 4’C). 7. Aspirate the supernatant cautiously but completely (the pellet may come off the walls in flakes). Resuspend pellet in 25 mL 1X TE buffer (this may take up to 2 h if yield is very high). When completely resuspended, add 25 g CsCl (see Note 3). 8. Add 2.0 mL ethidium bromide (10 mg/mL in HzO: Caution-toxic and carcinogenic). Centrifuge samples in 50-mL disposable tubes (3000g for 15 min at room temperature). 9. Use a VT150 (Beckman [Fullerton, CA]) or equivalent vertical rotor (a fixedangle rotor may be used, but may not yield optimal results) Fill a clear, thinwalled centrifuge tube with the supernatant of low speed spin, add CsCl-solution to fill tube, balance, and cap tube. Centrifuge for 24-36 h at 200,OOOg at 25°C. 10. Uncap or pierce holes in top of tube. Piercea hole mto the tube 0.5 cm below the lower band. Extract it with needle opening facing band under UV light (UV light necessary only if DNA yield is low). This should result m a 2--4-n& sample of the lower band only (avoid upper band containing genomic DNA). 11. Add 0.2 mL ethidium bromide to Quickseal tubes for a VT165.2 (Beckman) or equivalent rotor. Add sample and fill the tube with CsCI-solution. Following balancing and capping the tube, it is centrifuged for at least 4 h at 400,OOOg at 25% 12. After centrifugation, the DNA is removed by inserting a syringe -0.5 cm below band, as described in step 10, and a yield of l-2 mL should be expected. The upper band should be invisible or visible only under UV light. 13. Contaminating ethidium bromide should be removed by extraction with saltwater saturated isopropanol until supernatant is completely clear. The DNA-con-

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taming sample should be dialyzed against a > loo-fold excess of TE with at least one change of the dralysrs buffer. 14. Measure OD,,, and OD,,, in an aliquot (- 1:50 dilutton of DNA in 1X TE). 1 OD,,, unit = 50 c(g double-stranded DNA/ mL. The OD,,,,/OD,,, ratro should be 1.85-l .95. 15 The identity, quality, and especrally the absence of genomrc DNA of the plasmrd should be checked by restriction analysis. The yield varies with plasmid and vector between 2 and 20 mg, startmg from a 200-mL culture.

3.2. Transfecfion and Transient Expression of 293 Cells This procedure is performed using 16-cm Petri dishes. The method can be scaled down to different-sized dishes, with volumes corrected by volume and DNA amount by plate surface area. 1 Trypsmrze confluent flasks of 293 cells (cells may be obtained from American Cell Culture Collectron). Dilute cells to 5 x 106/16 cm plate. The incubator CO2 concentration should be 5%. 2. Let cells grow for 40-72 h to cover 50% of plate surface area. (Cells should not be used if accelerated rates of growth are observed, this can often occur at or beyond passage 20.) To a disposable plastic tube that will hold 2 mL x number of plates to be transfected wrth a particular DNA, add -20 pg DNA (2X CsCl gradient purified) per plate. Optimal concentratron must be checked through experimentation 3. Sequentially add sterile water to a final volume of 1 ml/plate, then add 2MCaQ (125 &/plate), and finally add 1 mL of 2X BBS/plate, mix thoroughly, and watt for >90 s (see Note 4) 4 Gently add 2 mL of the solution to the plate, carefully avoiding dislodging the cells; the media should turn from orange to yellow at the point of addition. MIX gently by slightly tilting the plate back and forth. Do not take more plates from the Incubator than you can handle m 3 min. 5. Place plates m a 3% CO2 incubator for 24 h. After this, remove media and gently add 15 mL of 293 MEM medium Place plates in a 5% CO2 incubator Fortyeight hours from the time of DNA addmon, the cells should be optimal for harvesting (see Note 5). Check viability of the cells. Some receptors may be toxic to them (see Note 6).

3.3. Membrane

Preparation

1. Wash plates once with 5 mL prewarmed PBS. Rinse cells from plate surface with 8-10 mL PBS/plate

2. Transfer the cell suspensionto a centrifnge tube for low speed centrifugation. Spin down cells at low speed (300g for 5 mm). Aspirate PBS. (For a crude membrane preparation, proceed to step 5 ) 3, Homogenize cell pellet in 1 ml/plate of mannitol buffer by 20 strokes in a glassglass homogenizer (e.g., Tenbroek-type) Centrifuge homogenate for 5 mm at

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10008. Aspirate supernatant carefully into a fresh tube, resuspend pellet in 0.5 mL/ plate of mannitol buffer by 10 strokes in a Tenbroek homogenizer. Repeat centrifugation step. Combine second with first supernatant; discard pellet 4. Centrifuge pooled supernatant at 40,OOOg for 30 min. 5, Homogenize the cell pellet using a tissue homogemzer (Ultra-turrax [IKA, Staufen, Germany] or Polytron [Brinkman, Westbury, NY]) at 90% of top speed for 15 s in -10 times the pellet volume of hypotonic buffer (e.g., 10 mA4 TrisHCl, pH 7.5). Centrifuge at 23,000g for 20 min and aspirate supernatant. 6, Homogenize membrane pellet in appropriate volume of buffer, depending on transfection efficiency and following experiments (-5 s 90% top speed with the tissue homogenizer), Repeat homogenization and centrifngation step as often as necessary, depending on object and type of analysis (e.g., to remove glutamate or other neurotransmitters from membranes as well as antagonists eventually added during cell culturing).

4. Notes 1. 2. 3. 4.

Volumes m the DNA Maxi-Prep Procedure can be scaled up or down accordingly. Vigorous mtxmg would lead to excess release of genomic DNA. Weigh out and adjust volume and CsCl if other than Quickseal centrifuges are used. With some BBS solutions, a precipitate may form more rapidly; in such cases, shorten the time. The precipitate will form m most solutions if you wait long enough The first sign of precipitate formation is as a very faint haze m the solution, which can only be seen in a clear polycarbonate type tube. If the precipitate can be seen under the microscope immediately after the solution is added to the cell growth media, shorten the time, or make new 2X BBS, as the subsequent yield may be low. 5. The optimal time point may vary with the protein m question, but harvesting after 48 h is a good starting point. 6. The addition of functional antagonists of the nascending protems may be essential, if the function interferes with the viability of the cells, e.g., by promotmg excess entry of Ca*+.

Acknowledgments The authors would like to thank Heidi Schott for expert secretarial help. The financial aid by the Deutsche Forschungsgemeinschaft, the N. M. F. Z., and the Strftung Rheinland-Pfalz fiir Innovation is also gratefully acknowledged.

References 1. Kein&ren, K., Wisden, W., Sommer, B., Werner, P., Herb, A., Verdoorn, T. A., Sakmann, B , and Seeburg, P. H. (1990) A family of AMPA-selective glutamate receptors. Sczence 249,556-560. 2. Ltiddens, H., Pritchett, D. B., Kbhler, M., Killisch, I., Keinanen, K., Monyer, H., Sprengel, R., and Seeburg, P. H. (1990) Cerebellar GABA* receptor selective for a behavioural alcohol antagonist Nature 346,648-65 1.

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3. Matthew, P. A., Mason, J. I., Trant, J. M., and Waterman, M. R. (1990) Incorporation of steroidogenic pathways which produce cortisol and aldosterone from cholesterol into nonsteroidogenic cells. Mol. Cell Endocrinol. 73, 73-80. 4. Wei, L., Alhenc, G. F., Soubrier, F., Michaud, A., Corvol, P., and Clauser, E. (199 1) Expression and characterization of recombinant human angiotensin I-converting enzyme: evidence for a C-termmal transmembrane anchor and for a proteolytic processing of the secreted recombinant and plasma enzymes. J. Bzol. Chem. 266,5540-5546.

5. Nakai, A., Sakurai, A., Macchia, E , Fang, V., and DeGroot, L. J (1990) The roles of three forms of human thyroid hormone receptor m gene regulation. Mol Cell Endocrinol 72, 143-148. 6. Chen, C. and Okayama, H. (1987) High-efficiency transformatton of mammalian cells by plasmid DNA. Mol. Cell Blol. 7,2745-2752. 7 Behr, J. P., Demeneix, B., Loeffler, J. P., Perez, and Mutul, J. (1989) Efficient gene transfer into mammalian pnmary endocrine cells with lipopolyamine-coated DNA. Proc Natl. Acad SCL USA 86,6982-6986. 8. Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. (1987) Lipofectton: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl Acad. Sci. USA 84,7413-7417. 9. Felgner, J. H., Kumar, R., Sridhar, C. N., Wheeler, C J., Tsai, Y. J., Border, R., Ramsey, P., Martin, M., and Felgner, P. L. (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol. Chem. 269,2550-2561.

10. Neumann, E., Schaefer Ridder, M., Wang, Y , and Hofschneider, P. H (1982) Gene transfer into mouse lyoma cells by electroporation m high electric fields. EMBO J 1,841-845.

11. Johnston, S. A. (1990) Biolistic transformation: microbes to mice. Nature 346, 776,777. 12. Cutting, G. R., Lu, L., Ohara, B. F., Kasch, L. M., Montrose-Rafizadeh, C., Donovan, D. M., Shimada, S., Antonarakis, S. E., Guggino, W. B., Uhl, G. R., and Kazazian, H. H. (199 1) Cloning of the y-aminobutyric acid (GABA) p 1 cDNA: a GABA receptor subunit highly expressed in the retina. Proc. Natl. Acad, Sci. USA H-$2673-2677.

13. Seeburg, P. H., Wisden, W., Verdoorn, T. A., Pritchett, D. B., Werner, P., Herb, A., Ltiddens, H., Sprengel, R., and Sakmann, B. (1990) The GABA* receptor family: molecular and functional diversity. Coldspring Harbour Symp. Quant. Biol 55,29-44.

14. Ltddens, H. and Wisden, W. (1991) Functron and pharmacology of multiple GABA, receptor subunits, Trends Pharmacol. Sci. 12,49-5 1. 15. Sieghart, W. (1995) Structure and pharmacology of y-aminobutyric acid* receptor subtypes. Pharmacol. Rev. 47, 18 l-234. 16. Schofield, P. R., Darlison, M. G., Fujita, N., Burt, D. R., Stephenson, F. A., Rodriguez, H., Rhee, L. M., Ramachandran, J., Reale, V., Glencorse, T. A., Reale, V.,

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18.

19.

20.

21. 22.

23.

24.

25.

63

Seeburg, P. H., and Barnard, E. A. (1987) Sequence and functional expression of the GABA, receptor shows a ligand-gated receptor super-family. Nature 328, 22 l-227. Levitan, E. S., Schofield, P. R., Burt, D. R., Rhee, L. M., Wisden, W., Kohler, M., Fujita, N., Rodriguez, H. F., Stephenson, F. A., Darlison, M. G., Barnard, E., and Seeburg, P. H. (1988) Structural and functional basis for GABA* receptor heterogeneity. Nature 335,76-79 Pritchett, D. B., Sontheimer, H., Shivers, B. D., Ymer, S., Kettenmann, H., Schofield, P. R., and Seeburg, P. H. (1989) Importance of a novel GABA* receptor subunit for benzodrazepine pharmacology. Nature 338,582-585. Sigel, E., Baur, R., Trube, G., Mbhler, H., and Malherbe, P. (1990) The effect of subunit composition of rat brain GABA* receptors on channel function. Neuron 5,703-711. Perez Velazquez, J L. and Angelides, K. J. (1993) Assembly of GABA* receptor subumts determines sorting and localization m polarized cells. Nature 361, 457-460. MacDonald, R. L. and Olsen, R. W. (1994) GABA* receptor channels. Annu Rev. Neurosci. 17,569-602. Shivers, B. D., Killisch, I., Sprengel, R., Sontheimer, H., Kohler, M., Schofield, P. R., and Seeburg, P. H. (1989) Two novel GABA, receptor subumts exist m distinct neuronal subpopulations. Neuron 3,327-337 Moody, E. J., Suzdak, P. D., Paul, S. M., and Skolnick, P. (1988) Modulation of the benzodiazepinely-aminobutyric acid receptor chloride channel complex by inhalation anesthetics. J. Neurochem. 51, 1386-1393. Korpi, E. R. and Ltiddens, H. (1993) Regional y-aminobutyric acid sensittvrty of t-butyl-bicyclophosphoro[35S]thionate binding depends on y-ammobutyric acid*receptor a subunit. Mol. Pharmacol 44,87-92. Ltiddens, H., Seeburg, P. H. S., and Korpi, E. R. (1994) Impact of p and y variants on ligand binding properties of y-aminobutyric acid type A receptors. Mol. Pharmacol.

45,810-814.

5 The Generation

of Receptor-Selective

Antibodies

John J. Mackrill 1. Introduction Antibodies have played a key role in receptor research. These immunoglobulms (Ig) frequently have greater affinities and selectivities for receptors than pharmacological probes. They are often the only probes available that can discern proteins with similar structural and pharmacological properties, such as distinct isoforms or splice-variants of a receptor family. Furthermore, antibodies may be utilized to determine a wide range of a receptor’s characterrstrcs: its apparent molecular mass by immunoblotting; its interaction with other proteins, covalent modifications, and turnover rates by immunoprecipitation; its localization in tissues, cells, and subcellular domains by immunohistochemistry, immunocytochemtstry, and immunoelectron microscopy; its primary structure by means of screening complementary DNA (cDNA) expression libraries; and its functional properties by virtue of some antibodies acting as receptor agonists/antagonists. An antigen is any substance that can elicit production of antibodies. When immunized into a host animal, antigens are also referred to as immunogens. Antigens may contain one or many epitopes (i.e., moieties that may form an antibody binding site). Two broad classes of antibodies may be defined. Polyclonal antibodies (PAbs) are derived from the sera of animals immunized with either the whole or part of the target receptor’s amino acid sequence, or the purified native receptor. Consequently, they contain a population of antibodies with a range of affinities and selectivities for the antigen. Monoclonal antibodies (MAbs) consist of one type of antibody molecule, of single affinity and selectivity for the antigen. Monoclonal antibodies may be generated by clonal selection of hybridomas between immunoglobulin-deficient B-cell lines and splenocytes derived from a host animal (normally rats or mice) immunized From

Methods In Molecular Bology, vol 83 Receptor Signal Transduction EdIted by R A J. Chalks Humana Press Inc , Totowa, NJ

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Protocols

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with the antigen (1,2). Recently, monoclonal antibodies have been generated using recombinant DNA techniques (3); however, use of such techniques is beyond the scope of this chapter. Monoclonal antibodies have a number advantages over polyclonal antibodies: MAbs are of single affinity, which makes them particularly useful in functional studies; once a clonal hybridoma line is established, the supply of antibody is unlimited compared with PAbs, the availability of which is limited by the volume of serum from a terminal bleed of the immunized animal; and background signals in immunoassays tend to be lower when using MAbs compared to PAbs. However, PAbs frequently give higher signals in immunoassays;production and screening of MAbs is more labor intensive and expensive than for PAbs; and cell-culture facilities are required for MAb production. Generation of PAbs is within the scope of most laboratories engaged in receptor research; furthermore, commercial services are available for synthesis of peptide antigens, immumzation of host animals, and preparation of antisera. Two strategies for the production of receptor-selective PAbs are described. One uses synthetic peptides derived from the primary sequence of the receptor as an immunogen (4). The other employs bacterially-expressed fusion proteins, in which part of the receptor’s amino acid sequence is synthesized by a bacterial host, in frame with a protein tag, such as glutathione-S-transferase, which assists in its purification. The fusion protein approach is cheaper than the synthetic peptide strategy, and since the amino acid sequence expressed is usually larger, fusion proteins tend to be more immunogenic and to resemble the native conformation of the target. However, use of synthetic peptides as antigens is less time-consuming and also permits production of antisera against a small region of the target receptor, increasing the probability of obtaining highly specific, site-directed probes. Ideally, a combination of approaches, against a number of different regions of the receptor, should be employed.

2. Materials 2.1. Reagents All reagents were from Sigma-Aldrich (Dorset, UK), except where noted. 1. Phosphate buffered saline (PBS): 8 g/L NaCl, 0.2 g/L KCI, 1.44 g/L Na*HPO,, K&PO,, adjustpH to 7.2-7.4 with HCI, if necessary. PBS/T:0.2% (v/v) Tween-20 m PBS; m-PBS: 5% (w/v) nonfat milk (Marvel or equivalent) m PBS; m-PBS/T: 5% (w/v) nonfat milk in PBS-T.

2. 4X separatinggel buffer: 1.5MTris-HCl, pH 8.3; 4X stackingbuffer: 0.5MTrisHCl, pH 6.8; 30% acrylamide stock (Ultrapure Protogelw, 30% [w/v] acrylamide,

0.8% [w/v] bisacrylamide [37.5:11, from National Diagnostics, Atlanta, GA).

Generation of Receptor-Selective

3.

4.

5 6.

7. 8. 9. 10 11. 12. 13. 14.

15. 16. 17.

18.

pAbs

67

Caution: Acrylamide is a cumulattve neurotoxin, and so it is essential to wear gloves whenever it is handled. Separating gel mix: 2 66 mL of 30% acrylamide stock, 2.5 mL of 4X separating buffer, 100 pL of 10% (w/v) sodium dodecyl sulfate (SDS), and 4 705 mL of distilled, deionized water. Add 10 pL of TEMED (N,N,N’,N’-tetramethylethylene diamine) and 25 pL of 10% (w/v) ammonium persulfate (APS) just prior to pouring the gel. Stackmg gel mix: 0.833 mL of 30% acrylamide stock, 1.25 mL of 4X stackmg buffer and 50 pL of 10% (w/v) SDS. Add 20 pL of APS and 5 pL of TEMED just before pouring. 5X SDS-PAGE sample buffer (10 rnL): 1.25 mL 1M Tris-HCl, pH 6.8, 4 mL 10% SDS, 1 mL 1% (w/v) bromophenol blue, 2 mL glycerol, 10 mA4 dithiothreitol. Dissolve, warming in a 37°C water bath ifnecessary, then make up to 10 mL with distilled, deionized water. Store at -2O’C. SDS-PAGE running buffer: 30.2 g Tris, 188 g glycine, 10 g SDS in 10 L of distilled, detonized water. Blottmg buffer: 150 mM glycine, 20 mM Tris, 0.037% (w/v) SDS, 10% (v/v) methanol. (The pH of this solution should be -8.3, though it does not usually have to be adjusted ) Nttrocellulose membranes, such as Protran mtrocellulose (Schletder and Schuell, Dassel, Germany). 10X Ponceau S: 2% (w/v) Ponceau S, 5% (v/v) acetic acid. Coomasste stain: 0.25% (w/v) Coomassie brilhant blue R250 in 50% (v/v) methanol and 10% (v/v) acetic acid; destain: 25% (v/v) methanol and 7% (v/v) acetic acid. Horseradish peroxidase (HRP) conjugated antirabbit/guinea pig IgG antisera (Sigma-Aldrich, Dorset, UK). Enhanced Chemiltinescence (ECL) reagents (Amersham, Buckinghamshire, UK). Coating buffer 10 mM NaHCO, pH 9.0. OPD solution: 40 pg/mL o-phenylene dramine (Sigma) m 100 mM sodium acetate, pH 5. Sulfosuccimmyidyl 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (sulfoSMCC) and Excellose GF-5 Desalting Columns (from Pierce and Warriner, Cheshire, UK). 10 mM sodium phosphate buffer, pH 7.2: 6.84 rnJ4 Na2HP04, 3.16 rnA4 NaH,P04. Protein A conjugated to agarose (such as Protein A Superose from Pharmacra, Uppsala, Sweden). Activated chromatography beads, such as Affigel 10 (Bio-Rad, Hertfordshire, UK), Tosyl Activated Agarose (Pierce & Warriner), or CNBr-activated Superose (Pharmacia). Elution buffer A: 100 mM Tris-HCl, 1 mM MgSO,, 0.1 mA4 ZnClz, pH 7.0; elution buffer B: 100 m&f Tris-HCl, pH 8.0; elutton buffer C: 100 mM glycine, pH 2.5; elution buffer D: 100 mM triethylamine, pH 11.5 (freshly prepared); elution buffer E: 50 mM NaCl, 100 mA4 Tris-HCl, pH 8.0; neutralization buffer A: 1M Trns-HCI, pH 8.0, neutrahzation buffer B: 10 mM Tris-HCl, pH 8.8

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2.2. Apparatus I. For ELISA: 96-well PVC plates (NUNC A/S , Rosktlde, Denmark). Microplate Reader, such as Bio-Rad Model 450. 2. For immunoblottmg: Protean (Bio-Rad) SDS-PAGE minigel apparatus or equivalent; Atto semidry blotting apparatus (Atto, Tokyo, Japan) or equivalent; power supply capable of generating an output of at least 400 mA and 300 V (such as PowerPac 300 from Bio-Rad); 3MM filter paper (Whatman, Kent, UK). 3. For chromatography* disposable plastic chromatography columns (l-10 mL volume), such as Econo-Pat columns (Bio-Rad), Pierce Disposable Plastic Columns (Pierce & Warriner), or similar. 4. Microconcentrators: with 30-kDa cutoff membranes, such as Centricon and Centriprep C30 devices (Amtcon, Beverly, MA). 5. GRI AX X-ray film or similar and film cassettes (Genetic Research Instrumentation, Essex, UK). 6. Standard benchtop centrifuges and microfuges.

3. Methods

3.7. Design and Synthesis of Synthetic Peptides The features of an amino acid sequence that make good immunogen have

been determined mainly by empirical methods. Although antibodies are capable of recognizing single amino acids, or moieties within single molecules, epitopes within proteins are usually greater than 6 amino acid residues long. Ideally,

synthetic peptide antigens should be greater than 15 ammo acids long;

above 25 residues, the cost and efficiency of synthesis become prohibitive. Peptide antigens should be hydrophilic, flexible, and will ideally represent a surface-exposed sequence within the native protein. Antigenicity can be predicted using algorithms that select for such features, for example, the algorithm of Jameson and Wolf (5). Although such algorithms are far from infallible, they are often included in computer software packages for sequence analysis (see Note 1). Such programs have an advantage because candidate peptide antigen sequences can be compared to sequences in protein databases (for example, EMBL and GenBank databases), which enables prediction of unwanted crossreaction of antipeptide antibodies with proteins bearing a similar amino acid sequence. In receptor proteins, ammo-(N)- and carboxy-(C)termini, as well as mtra- and extracellular loops, are generally antigenic. Once a suitable peptide sequence has been selected, several custom modifications may be made. If not present, a cysteine residue is usually incorporated on N-terminus, in order to facilitate conjugation to a carrier molecule. Cysteine residues within the peptide sequence may be replaced by serine residues, in order to prevent formation of intramolecular disulfide bridges. Two or three glycine residues may be added between the end of the selected sequence and

Generation of Receptor-selective

pAbs

69

the terminal cysteine. These act as spacers, increasing the distance of antigen from its site of conjugation to the carrier, thereby increasing its flexibility and accessibility. Peptide synthesis is beyond the budget of most laboratories engaged in receptor research. Consequently, commercial or in-house services are normally employed. Synthesis is usual performed by fluorenylmethoxycarbonyl-(Fmoc)polyamide solid support chemistry (6). Synthesis at 0.25 mm01 scale is sufficient for immunizing several host animals and screening antisera. Ideally, the purity of the peptide should be checked by mass spectroscopy and high-pressure liquid chromatography. It should be supplied m a lyophilized, fully reduced form. The cost and quality of commercial peptide synthesis varies widely, so it is worth shopping around before placing an order.

3.2. Production of Fusion Profelns Fusion proteins are usually produced by overexpression in bacterial systems. As mentioned, they consist of the amino acid sequence of interest expressed in frame with a tag that assists purification. Two commonly used tags are glutathione S-transferase and hexahistidine. Overexpression of fusion proteins consists of isolation of a cDNA encoding the required amino acid sequence (often by reverse transcription-polymerase chain reaction from an appropriate cDNA library), its cloning mto a suitable expression vector in the correct orientation, transfection into a suitable expression system, induction of overexpression, and isolation of the overexpressed protein. Detailed protocols on expression vectors are usually supplied with overexpression systems, or may be found in literature dealing with molecular cloning approaches (7,14). The size of a receptor-derived amino-acid sequence that may be expressed as a fusion protein is less restrictive than that synthesized as a peptide. Most commercially available expression vectors allow the production of fusion proteins containing at least 400 amino acid residues of the receptor sequence. For many receptors, this permits expression of the entire primary sequence. If a particular region of the receptor is required, because of similarity with other proteins, or if site-directed antibodies are desired, features that make an amino acid sequence potentially antigenic apply equally well to the design of fusion proteins as they do to synthetic peptides.

3.3. Conjugaflon of immunogens to Carriers Conjugation of antigens to carrier proteins, such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA), is usually required for the generation of high-titer (high-affinity) antisera against peptides or fusion proteins of less than 40 amino acid residues. Although preactivated carrier proteins are commercially available, it is useful to couple the antigen to two distinct carri-

70

Mackrill

em, using bifunctional crosslinkmg agents. One antigen-carrier conjugate may then be used for immunizations and the other for screening and affinity-purifying the resulting antibodies, m order to avoid problems associated with anticarrier protein immunoreactivity. A variety of bifunctional crosslinking agents may be employed for the conlugation of a peptide to a carrier. Many conjugation protocols require that the peptide/fusion protein is fully reduced and has a cysteine residue at the couplmg site, usually at its C-terminus. A typical conjugation protocol is given below: 1 All steps should be carried out in glassware, since peptides, proteins, and conjugates tend to stick to plastics. Dissolve 8 mg of KLH or BSA in 500 l.rL of 10 mM sodium phosphate buffer, pH 7.2. 2. Add 2 mg of sulfa-SMCC. Incubate for 1 h at 30°C with constant stirring 3 Separate the carrier-SMCC conjugate from the other reagents by passing down an exocellulose GF-5 column pre-equilibrated with elution buffer A. Elute usmg the same buffer collect m 1-mL fractions. Monitor for protein by measurmg

absorbanceat 280 nrn. 4. Mix 4 mL of the A 280nmpeak with 8 mg of the reduced synthetic peptide Incu-

bate overnight at 4°C with continuousstirring. 5. Store the conjugate at -20°C in glass vials.

3.4. Immunization Protocols Rabbits and guinea pigs are most commonly used for the production of polyclonal antisera. Ideally, 3-4 animals should be immunized with each antigen, in order to increase the probability of obtaining high-titer antisera. A prennmunization test bleed should be taken for use as a negative control in immunoassays. Two immunization routes are commonly employed in rabbits and guinea pigs. Intravenous mjectton of the antigen is unsuttable for primary immunizations, but may be used to elicit high-titer antibody production with booster immunizations. However, there is a risk of anaphylactic shock with intravenous immunizations, with loss of the immunized animal. With subcutaneous immunizations, the antigen is often mixed with an adjuvant, a substance that nonspecifically stimulates immune response (8). Typical adjuvants are Freund’s Complete Adjuvant (FCA, for primary immumzations) and Freund’s Incomplete Adjuvant (FIA, for booster mnnunizations). Freund’s Adjuvants consist of mineral oil with (FCA) or without heat-killed Mycobacterium tuberculosu. The action of adjuvants are twofold: first, they deposit the antigen m a form that is slowly cleared from the host (an emulsion in the case of FCA/FIA); second, they contain substances that stimulate the host’s immune system (heat-killed bacteria m the case of FCA). These properties serve to enhance the immunologic response of the immunized animal against the injected antigen.

Generation of Receptor-Selective pAbs

71

A typical schedule for the subcutaneous immunization of rabbits would consist of injection of 100 ~18of antigen in 500 pL of PBS, mixed with 500 & of FCA (by passing between syringes joined by a Luer fitting), into five different sites per host animal; booster immunizations would follow the same protocol, replacing FCA with FIA, 28 d after the previous immumzation; up to three boosts would be given, Test bleeds would be taken for immunoassay 14 d after each immunization; once the titer of the serum is sufficiently high, or 28 d after the final boost, the immunized animals should be bled out by cardiac puncture, under terminal anesthesia. It essential to obtain the appropriate licensing and training before immunizing and bleeding animals (see Note 2). Alternatively, immunization and bleeds of animals may be provided as an in-house service rn universities and research establishments. It is also important to note that immunization schedules (number of animals, immunization routes, and so on) may be limited by licensmg and by the schedules favored by in-house services. 3.5. Preparation of Antisera Clotting provides a means to separate crude antisera from whole blood. Although there are devices commercially available for this purpose, the following protocol is inexpensive and reliable: 1. Incubate freshly obtained bleeds in I-mL aliquots for 1 h at 37°C m 1.5mL Eppendorf tubes. 2. Move the clotted material from the sides and push tt toward the bottom of each tube, using a cocktail stick. 3. Incubate overnight at 4°C to contract the clot. 4. Centrifuge at 1000gmax for 10 min in a microfuge. 5. Collect the supematant, taking care not to dtsturb the pellet. Store the majorrty of the supematant (antiserum) in 500~pL aliquots at -2O’C. For short-term use, store the serum at 4°C in the presence of 0.02% (w/v) sodmm azrde.

3.6. Screening

of Antisera

3.6.7. Enzyme-Linked Immunoassay (ELBA) Screening ELISA is a sensitive, semiquantitative assay for immunoreactivity of antibodies against a chosen antigen, Antibody-capture ELISAs are frequently used to assessthe immunoreactivity of antisera (9): 1. Coat 96-well polyvinylchloride plates with 1 pg/well of synthetic peptide/fusion protein in 100 @,/well of coating buffer. 2. Incubate for 2 h at room temperature, or overnight at 4”C, m a humid, sealed box. 3. Remove the antigen solution by flicking the plate and leave it draining on paper towels. (As an alternative, antigen solution can be collected and stored at -20°C

72

4. 5.

6. 7

8. 9. 10.

Mackrill if the supply of peptide/fusion protein is limited. Since the coating buffer contams a large excess of the antigen, to ensure rapid binding to the PVC wells, the same solutton can be used to coat one or two more plates.) Block the remaining sites for protein binding with 100 pL/well of m-PBS/T for 1 h at room temperature. Incubate duplicate wells with 100 @.,of serral dilutions of the antiserum m m-PBS/T. A typical dilution range for a polyclonal antiserum would be from 1:50 to 1:25,600. Several wells should be incubated with m-PBS/T alone as negative controls. Leave for 2 h at room temperature. Remove the primary antiserum solution by flicking the plate. Wash with three changes of PBS/T over 15 min. Incubate for 1 h with 100 @/well of a suitable dilution (usually -1:2000) of a horseradish peroxidase (HRP)-conjugated secondary antibody, which ~111recognize the primary antiserum tested (i.e., antirabbit IgG(HRP) for a rabbit pnmary antiserum). Remove the secondary antibody Wash wells for 2 x 5 mm with PBS/T, then once for 5 min with PBS Add 100 @,/well of OPD solution. Incubate for 10-30 min at room temperature. Stop the reaction with 50 @well of 1M sulfuric acid. Absorbance of the soluble orange reaction product should be measured at a wavelength of 492 nm, on an ELISA plate reader. Wells mcubated with no primary antibody should be subtracted, as a background, from other values

3.62. lmmunoblot Screening Immunoblot screening of antisera is important in order to ensure that they are recognizing a target protein of appropriate apparent molecular mass. There are several steps in immunoblot assays. 3.6.2.1.

SDS-PAGE

1. Set up SDS-PAGE apparatus according to the manufacturer’s instructions. For most receptor proteins, a 0.75-l-mm-thick, 8% SDS-PAGE minigel will provide adequate resolution. 2. For two 0.75~mm-thick mimgels, make 10 mL of separating gel. Pour the gel into the apparatus, leaving about one-fourth of its total height for the stacking gel. Pour the gel slowly to avoid creatmg bubbles, since these Inhibit polymerization Slowly overlay each gel with -100 pL of water-saturated (70%) isopropanol. 3. Once the separating gels have set (less than 60 min), wash off the saturated isopropanol with distilled, detonized water. Make up 5 mL of separating gel. Pour the stacking gel into the gel cassette, to the top of the plate. Insert the sample combs at an angle of -30° to horizontal. This ensures that no bubbles are trapped underneath each tooth of the comb, which would otherwise cause uneven setting of the gel, leading to uneven bands. Then adjust the combs so that they are horizontal. Allow the stacking gels to set for about 30 min.

Generation of Receptor-Selective pAbs

73

4. Add 5 pL, of 5X SDS-PAGE sample buffer to each of 20-pL samples of receptorbearing membranes, fusion proteins, or purified receptor proteins (see Note 3) in Eppendorf tubes. Positive and negative control samples should be included. Prestamed or unstained mol wt standards should also be run on the same gel, in order to estimate apparent molecular masses. Make a small hole m the lid of each tube, using a needle. Incubate samples at 95’C for 3 min, then centrifuge at lO,OOOg,,, for 20 s. 5. Remove the sample combs from the gels. Assemble the gel apparatus for electrophoresis, according to the manufacturer’s instructions. Fill the upper tank with SDS-PAGE running buffer, above the level of the sample wells. 6. Load the samples into appropriate wells, underneath the running buffer. Ideally, long, narrow pipet tips should be employed. Assemble the apparatus and electrophorese according to the manufacturer’s instructions. An 8% SDS-PAGE minigel should be run at about 15 mA constant current per gel Under these conditions, the bromophenol blue dye-front will be within -1 cm of the end of the gel within an hour.

3.6.2.2. WESTERN

BLOTTING

1. Soak four sheets of Whatman 3MM filter paper and one sheet of nitrocellulose, all cut to the same drmensrons as the gel, m blottmg buffer. 2. Place two sheets of the filter paper, one on top of the other, onto the anode of a semidry Western blottmg apparatus. Place the sheet of nitrocellulose on top of these. Sheets of soaked filter paper/nitocellulose should be placed onto the sandwich in a U shape, the edges being evenly lowered onto the layer below. This prevents bubbles being trapped between layers, which would otherwise inhibrt effictent transfer 3. Remove the gel on which the test samples have been resolved and carefully place it on top of the nitrocellulose. 4. Place another two layers of buffer-soaked filter paper over the gel. 5 Assemble the apparatus. For most target proteins, electrophoresis at 0 65 mA/cm2 of gel for l-2 h is adequate to ensure sufficient transfer (see Note 4).

3.6.2.3. IMMUNOSTAINING 1. Remove the nitrocellulose sheet from the transfer apparatus. Transfer to a clean plastic tray. In order to verify efficient transfer of proteins, stain the blot with Ponceau S diluted in deionized, distilled water Wash once with deionized, distilled water and mark the positions of mol wt markers, lanes, and proteins of interest, using a ball-point pen. Completely remove the stain by extensive washmg with deionized, distilled water. 2. Incubate in m-PBS overnight at 4’C with constant shaking. 3. Remove the m-PBS and replace with the antiserum to be tested, diluted m the same solution, in a vol of 200 @/cm2 of nitrocellulose. 4. Incubate for 1S-3 h at room temperature, with constant shaking.

74

Mackrill

5. Wash for 3 x 10 mm with PBS. Incubate with a 1:2000 dilution of an appropriate secondary antibody-HRP conjugate m m-PBS (200 &/cm2), with constant shaking 6. Wash for 3 x 10 min with PBS. Develop the blot using an Enhanced Chemiluminescence (ECL) kit, according to the manufacturer’s instructions (see Note 4). Expose to X-ray film

3.7. Purification of Antibodies The titer and specificity of antisera may be improved by purifying antibodies, or subsetsof antibodies, from the crude antiserum. The followmg methods purify either the immunoglobulin G (IgG) subtype of antibodies, or those recognizing the immunogen, from the crude antiserum.

3.7.1. Using Protein A-Agarose Protein A is a peptide from the cell wall of Staphalococcus aweus that binds IgG antibodies from various species with high affinity. When conjugated to a chromatographic support, protein A is extremely useful for isolating the IgG family of antibodies from a crude antiserum (10). Such purification procedures not only produce IgG of high purity, but also concentrate these molecules. Furthermore, the IgG subtype of antibodies tend to have the greatest affinity for the antigen. It should be noted that although protein A is extremely useful for isolating IgGs from rabbit and guinea-pig, it is not as smtable for use with sera from other hosts, such as rats and mice. This protocol can be scaled up to allow production of large quantities of antireceptor IgG: 1. 2 3. 4.

Add 10 mL of column buffer B to 1 mL of packed protein A beads. Centrifuge at 4OOg,,, for 5 min. Discard supernatant. Repeat steps 1 and 2 once. Incubate washed beads with 4 mL of packed beads with 4 mL of crude antiserum supplemented with 400 4 of neutralization buffer A. Incubate overnight at 4°C with constant agitation, preferably on an orbital shaker. 5. Pack the beads into a disposable plastic column. Wash with 10 mL of elution buffer B and 10 mL neutralization buffer A. Elute IgG molecules bound to the column with elution buffer D in 500 pL steps. Collect elutant m Eppendorf tubes containing 50 pL of neutrahzation buffer A. 6. Monitor absorbance of each fraction at a wavelength of 280 m-n (1 OD unit =

-0.8 mg IgG protein/ml). 7. Concentratefractions of peak absorbanceusing Centricon C30 devices, accordmg to the manufacturer’s instructions. 8. Store at 4°C with 0.02% (w/v) sodium azide and 1 mg/mL bovme serum albumin (BSA). Assay for immunoreactivity by immunoblot and ELISA

3.7.2. Using the h-munogen for Affinity Chromatography This procedure isolates antibodies recognizing the antigen from all other proteins in a crude antiserum. As with protein A affinity-chromatography, it

Generation of Receptor-Selective

pAbs

75

can be scaled up to increase quantities of purified antibodies. Unlike protein A chromatography, antigen-affinity chromatography is not useful for purifying MAbs, since these should bind a single epitope on, and be of single affinity for, the antigen: 1. Preactivated beads are commercially available for the attachment of antigens to a chromatographic support. Covalently link the peptide- or fusion protem-KLH/ BSA conjugate to such a chromatographic resin, according to the manufacturer’s instructions. Carbonyldiimrdazole-activated beads are unsuitable for this procedure, since the linkage formed with the antigen-conjugate 1sunstable at high pH Note that the peptide/fusion-protein should be conjugated to a different carrier from that used in the immunizations, in order to avoid adsorptton of anti-BSA or -KLH antibodies. 2. Figure 1 describes a protocol for purification of anti-peptide/-fusion protein antibodies on a small scale. One milliliter packed volume of antigen coupled beads will usually bind several mtlhgrams of specttic antibodies. 3. Pooled antibodies may be concentrated usmg Centnprep C30 devices at 4°C accordmg to the manufacturer’s instructions. hnmunoreactivity of the pooled, concentrated antibodies should be assayed by immunoblot and ELISA Purified antibodies may be stored at 4°C in the presence of 0.02% (w/v) sodium azide and 1 mg/mL BSA.

3.7.3. Affinity Purification of Antibodies on Protein Transfers Antireceptor antibodies may be purified by their affinity for the receptor, or receptor fragment-fusion protein, resolved by SDS-PAGE and transferred onto nitrocellulose (II). Although this technique cannot be readily adapted for largescale purifications, it is useful for simple, rapid isolation of antiantigen antibodies: 1. Prepare an SDS-PAGE gel as described in Section 3.6 2 Use a preparative sample combs in the stacking gels, alternatively, the same sample can be loaded into individual wells. 2. Load the gel with a sample enriched in the immunogen, such as the purified fusion protein (100 pg protem/gel), or the receptor of mterest (1 O-l 00 pg purified receptor protein or up to 500 pg of membrane protein/gel; see Note 3). Electrophorese and blot as described. 3. Stain the blot with Ponceau S and mark the position of the protein of interest, using a ballpoint pen. Block for 1 h in m-PBS. 4. Incubate the blot for l-2 h with 4 mL of a 1:50 dilution of the crude antiserum in PBS. The diluted antiserum solution can be incubated with one or two more blots, for an hour each, if sufficient immunogenfreceptor protein is available. 5. Cut out the band of interest using a scalpel blade, usmg the pen marks made earher as a guide. 6. Incubate the mtrocellulose strips with 2 mL of elution buffer C for 10 min at room temperature. Neutralize this solution with 200 pL of neutralization buffer A. Add 0.02% (w/v) sodium azide, 1 mg/mL BSA, and store at 4°C. Assay the titer of this purified antibody fraction by immunoblot or ELISA.

Mackrill

76 1 mL peptide-/fusion mamx

REGENERATE Wash

with:

protein

COLUMN:

1. 10 mL elution 4. 10 mL elution

buffet B: 2. 10 mL elution buffet D: 5. 15 mL elution

buffer buffer

C ; 3. 10 mL elution B

Add I mL of crude 10mLofPBS

circulate through

diluted co1

Wash

with

Elute

B:

in

antiserum

20 mL of elution

acid-sensitive

Collect &ant into a tube containing 1 mL of neutralisation buffer A Elute

antiserum

buffer

base-sensitive

pAbs

Wash buffer

pAbs

buffer

wth

with B

B then 20 II% of

10 mL of elution

buffer

elution

buffer

C

buffer

D

E

10 mL of elution

1 with

10 mL of elution

Collect eluant into B tube containing 1 mL of neutralisation buffer A. Pool with acid-sensitive pAbs and concentrate to less than 0.5 mL

Fig. 1. Purification of anti-peptide-/fusion gen affinity chromatography.

Wash column wth 15 mL of elut~on buffer B; store at 4 C I” the same buffer contammg 0.01 % thnnerosal

protein antibodies from antisera by anti-

4. Notes 1. A range of software is available for analysis of the sequences of biological molecules. Although these programs tend to be expensive, they are useful for a laboratory engaged in receptor molecular biology, not only for predicting potential antigenic regions, but also for identifying putative phosphorylation sites, modu-

Generation of Receptor-Selective

pAbs

lator binding sites, protease cleavage sites, and for predicting secondary structure. Such software often contains routines for DNA sequence analysis and primer design. If the cost of such software is prohibitive, many DNA/protein analysis algorithms are accessible on the Internet. 2. In the UK, application forms for licensing of animal procedures can be obtained from The Home Office, 50 Queen Annes Gate, London. SW1 9AT. Applications should include full details of the immunization schedule(s) to be used. 3. Fusion proteins are ideal for screening of antisera by both ELISA and immunoblot. Synthetic peptides, however, are only useful for ELISA screening. In both cases, the reaction of antibodies against the native protein must be verified. This involves specific recognition of the receptor from lysates or membranes prepared from sources enriched in the receptor, or of the purified receptor, on immunoblots. Sources enriched in receptor include tissues in which expression levels are naturally high, or transfected mammalian cell-lines. Crude lysates may be prepared from such sources by adding 10 vol ( 10 mL/g) of 1X SDS-PAGE sample buffer; passing the mixture through a 26-gage needle until a fine suspension is obtained; heating to 95OC for 3 min; centrifugation at lO,OOOg,,, for 10 min; and storage of the supernatant at -20°C until use in immunoblot assays. For greater enrichment, either purified membrane fractions (22) or purified receptor proteins can be prepared. However, preparation of membranes and purified proteins is beyond the scope of this chapter; methodologies may be found in the hterature covering the receptor of interest, or in texts dedicated to these techniques. 4. Although the electrophoresis, blotting, and immunostaining conditions described in Section 3.6.2. are suitable for most proteins, they may not be ideal for all receptors, particularly those that are exceptionally small, large, charged, hydrophobic, or low in abundance. Immunoblot assays can be optimized at a number of stages: a. SDS-PAGE: The concentration of acrylamide in SDS-PAGE gels may be varied in order to increase resolution of proteins within a certain molecular mass range. For proteins >200 kDa, use 5% gels; since these have reduced __ mechanical strength, they should be made in 1.5~mm-thick gel cassettes. For proteins of <40 kDa, use 15% gels. b. Blotting: Not all proteins will be adequately transferred under the conditions given (13). Very large proteins (>200 kDa) will tend to be retained within the gel; to avoid this, methanol should be removed from the transfer buffer (since it reduces the size of the pores in the gel), the blotting time should be increased (up to 2-4 h), and the transfer current should be increased (up to 4 mA/cm2). Care should be taken that extreme blotting conditions do not damage the semidry apparatus because of heating effects. Alternatively, transfer can be performed overnight at 4°C. Efficiency of transfer can be assessed by Coomassie blue staming the gel after blotting and staining another gel loaded and run in an identical manner, though not blotted. Coomassie blue stain for 2 h, then incubate in several changes of destain over an hour. Comparison of pre- and posttransfer gels will give an estimate of how well proteins of the molecular weight of interest have been transferred. For small proteins, both

78

Mackrill the transfer time and current should be reduced. For such proteins, there is a possibility of transfer through the mtrocellulose membrane. To assay for this, place two sheets of nitrocellulose, one on top of the other, then set up the semidry apparatus as usual After transfer, Ponceau S and immunostain both blots. If an excessive amount of the protein of interest is present on the second membrane, reduce blotting time and current further. Alternatively, other custom transfer buffers and conditions, tailored to suit a particular protein, may be found in the literature. c. Immunostaining: There are a number of variables in mununostaining a Westem blot: the dilution of the primary and secondary antibodies used, the time of mcubation m these antibodies, the duration of washing steps, and the concentration of protein on the blot. The protocol given in Section 3.6.2.3.) is suitable for most antisera. For optimization, first test a range of antiserum dilutions (twofold series from 1:50 to 1:2500) against a fixed concentration of the membrane proteins of interest (40 ug membrane protein/lane for a minigel). Lanes can be cut from Ponceau-stained nitrocellulose membranes, using a scalpel; alternatively, use a preparative mmigel (loaded with 400 pg of protein), then cut the nitrocellulose into 10 strips. Once an antiserum dilution that gives an optimal signal-to-background has been selected, test this dilution against a blot bearing a dilution series of the membrane protein of interest from 100 to 0.1 pg protein/lane. This dilution series will also indicate the concentratton range over which antibody binding 1s linear. If very weak signals are obtained, increase the primary antiserum concentration and incubation time, decrease the duration of wash steps (to 3 x 5 min), or carry out the immunostaining at 4’C with overnight incubation m the primary antiserum. Alternatively, purify the antibodies as described. Try using antipolyvalent Ig-HRP conjugates to probe for tmmunoreactivity, tf it is suspected that antireceptor IgGs are not present. If very strong signals with high backgrounds are obtained, decrease the primary antiserum concentration and incubation time, increase the duration of the wash steps (to 3 x 15 min), and decrease the concentration of protein on the blot. Again, antibody purification may be useful. If high backgrounds are still present, add 0.2% (v/v) Tween-20 to the blocking buffer, or try using 3% (w/v) BSA (IgG free) m PBS, instead of milk. Once optimal staining conditions have been established, the following controls should be performed to verify that the signals produced are specific: preimmune serum, and/or the antiserum preincubated with an excess of the immunogen (10 pg peptide or fusion protein/ml) for 1 h, should not recognize the receptor protein when using identical conditions to those used with the untreated antiserum; the antiserum should not recognize bands m negative control samples, such as sham-transfected cells, under conditions in which it recognizes receptors in positive control samples, such as receptor-transfected cells; ELISA titrations of antiserum against a fixed concentration of antigen should saturate.

Generation of Receptor-Selective

pAbs

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References 1. Kohler, G. and Mtlstein, C (1975) Contmuous cultures of fused cells secreting antibody of defined specificity. Nature (London) 256,495-497. 2. Goding, J. W. (1987) Monoclonal Antibodzes. Principles and Practice. 2nd ed. Academic, London 3. Winter, G., Griffths, A. D., Hawkins, R. E., and Hoogenboom, H R. (1994) Making antibodies by phage display technology. Ann. Rev Immunol 12,433-455. 4. Walter, G. (1986) Production and use of antibodies against synthetrc peptides. J. Immunol. Methods 88, 149-161 5. Jameson, B. A. and Wolfe, H. (1988) The antigenic index* a novel algorithm for predicting antigenic determinants. CABIOS 4, 18 l-l 86 6. van Regenmortel, M. H. V., Briand, J. P., Muller, S., and Platte, S. (1988) Synthetic Pepttdes as Anttgens. Elsevier, New York. 7 Sambrook, J., Fritsch, E. F., and Mamatis, T. (1989) Molecular Clontng A Laboratory Manual 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 8 Freund, J. (1956) The mode of action of mrmunologic adJuvants. Adv Tuberc Res. 7, 130-148. 9. Engvall, E and Perlmann, P (1972) Enzyme-linked immunosorbent assay, ELISA. III. Quantitatton of specific antibodies by enzyme-labelled anti-tmmunoglobulin in antigen-coated tubes. J. Immunol. 109, 129-135. 10. Ey, P. L., Prowse, S. J , and Jenkin, C. R. (1978) Isolation of pure IgG,, IgG2, and IgGZb immunoglobulins from mouse serum using protein A sepharose. Btochemtstry 15,429-436.

11 Smith, D. E and Fisher, P. A. (1984) Identtfication, developmental regulation, and response to heat shock of two antigenically related forms of a maJor nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of anttbodies usmg polypepttdes immobilised on nitrocellulose blots. J Cell Btol. 99,20-28. 12. Evans, W. H. (1992) Isolation and characterization of membranes and cell organelles, in Preparative Centrifugation. A Practtcal Approach (Rickwood, D., ed.), IRL, Oxford, pp. 233-270. 13. Beisegel, U. (1986) Review: protein blotting. Electrophorests 7, 1-18. 14. Riggs, P. (1992) Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds.), GreeneAssociateslWiley Interscience, New York.

6 In Situ Hybridization Judith C. W. Mak and Peter J. Barnes 1. Introduction Recent advances in molecular cloning and gene-expression techniques have led to the identification and characterization of many G protein-coupled receptors, including new subtypes and sub-subtypes of previously identified receptors. With the dramatic increase in the number of these receptors, the technique of in situ hybridization (ISH) has become an essential addition to the tools available for receptor research. The principle of ISH is simple and it is a powerful technique that provides extensive information for the detection and localization of specific mRNA to individual cells in histological sections.The technique mvolves the hybridization of target mRNA to a labeled nucleic acid probe, under appropriate conditions formmg stable hybrids, followed by visualization of the location of the probe. ISH has been used primarily for the localization of DNA sequences (Z-3). In recent years the technique has expanded to the localization of viral DNA sequences,mRNA, and chromosomal gene mapping (4,5). Four different types of nucleic acid probes can be prepared for use in ISH: double-stranded DNA probes (.5j, synthetic oligonucleotide probes (5), single-stranded DNA probes (6), and RNA probes (7). Double-stranded probes are less sensitive than singlestranded probes, since the two strands reanneal, thus reducing the amount of probe available for hybridization to the target mRNA (7). For a less abundant mRNA, the most popular are RNA probes, since they are single-stranded, form highly stable RNA-RNA hybrids, and have a constant probe size and no vector sequences, all of which favor increased sensitivity and consistency. In addition, following hybridization, the nonhybridized probe is readily removed by RNase digestion (s). Oligonucleotide probes are also used regularly because of their high specificity, despite having sensitivity significantly lower than that of From* Methods m Molecular Wology, vol 83 Receptor Sfgngnal Transducffon Edlted by R A J Chalks Humana Press Inc , Totowa, NJ

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Protocols

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RNA probes (9). This drawback can be overcome by using a mixture of oligonucleotides that are complementary to different regions of the target gene. The probes can be labeled with radioactive or nonradtoactive tags. In past years, nonradioactive probes based on biotin, digoxigenin, or fluorescein labeling have been produced (10-12). ISH is traditionally performed with radioactive labels. This is because these labels still offer the maximum sensitivity and robustness for this techmcally demanding technique. There are several labels that can be used and each of these has certain advantages. 32Pyields relatively short exposure times, but with a poor resolution. It is ideal for use in the optimization of new experimental systemsbecause of the quick results. At the other extreme, 3H offers excellent resolution but may require extremely long exposure times, and so has limited usage. The traditional compromise nucleotide has been 35S,which has medium resolution with acceptable exposure times, typically a few days to a few weeks, depending on the target level. Recently, 33Phas also become available, which has physical properties similar to 35S(13). The most widely used and sensitive detection method for radioisotopes is autoradiography using photographic emulsions. The first step is to optimize the methods in order to obtain sensitive and reproducible results. The type of fixation, the use of protemase K, addition of formamide in the hybridization buffer, and the hybridization temperature have major influences on the signal intensity. In the following sections, we will outline the method of ISH using radioactive-labeled RNA probes that we follow for the investigation of P-adrenoceptor and muscarinic receptor subtype mRNAs in lung (14-17). With the availability of transcription vectors, the desired probe sequence corresponding to the coding region of the gene is subcloned mto a suitable vector, such as pGEM-3Z, pSP72, or pBluescript, so that tt is flanked by two different RNA polymerase initiation sites, thus enabling either sense-strand (control) or antisense-strand (probe) RNA to be synthesized. The plasmid is linearized with a restriction enzyme so that plasmid sequences are not transcribed, since these will cause high background (Fig. 1). 2. Materials

2.1. In Vitro Transcription 1. 5X Transcription buffer: 200 mi’t4Tris-HCl, pH 7.5,30 mA4MgC12,10mM spermidine, 50 mA4NaCl. 2. 100wdithiothreitol (DTT). 3. RNasin (human placental nbonucleaseinhibitor): 40 U/& 4. 10mM of ATP, GTP, CTP, andUTP madeup separatelyin H20, pH 7.0. Nucleotide mixture: 2.5 mM each of ATP, GTP, and CTP (prepared by mixing 1 vol H,O with 1 vol each of the 10 mA4 ATP, GTP, and CTP stocks).

83

In Situ Hybridization In Situ Hybridization

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Fig. 1. Diagrammatic representation of the synthesis of sense-strand (control) and antisense-strand &R RNA probes, using the pGEM-3Z plasmid (&R-cDNA) containing SP6 and T7 RNA polymerase promoters.

5. 6. 7. 8.

lOO@?UTP. 1 mg/rnL. linearized plasmid template DNA in [35S]UTPaS: 10 mCi/mL (Amersham, UK). SP6 RNA polymerase, T7 RNA polymerase, 20 U/pL. These enzymes are very labile and freezer for minimal time. 9. DNase (RNase free): 1 J&IL. 10. Yeast tRNA: 10 mg/mL. 11. Sephadex G-50 in 10 mA4 Tris-HCl, pH 7.5,l Store solutions

l-6 and 8-l 0 at -20°C,

water or Tris-EDTA

(TE) buffer.

or T3 RNA polymerase: at 15should be out of the -2O*C deep

mikf EDTA, and 0.1% SDS.

and 7 at -70°C.

2.2. Fixation 1. O.lM phosphate-buffered saline (PBS): Dissolve 8.79 g NaCl, 0.27 g KH2P04, and 1.35 g Na2HP04 (anhydrous) in a liter of distilled water.

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2. 4% paraformaldehyde in PBS. Paraformaldehyde powder is dangerous to mucous membranes. When handling, avoid contact with eyes and wear gloves and a mask. To prepare the solution in a fume hood, warm PBS up to 65°C. Only then, with vigorous and continuous stirring, siowly add paraformaldehyde until the solution is clear If necessary, add 1OM NaOH dropwise until the solution clears. Cool, filter with filter paper, and use within 24 h. 3. Gelatin chrome-alum subbed slides: Place the glass slides in a slide rack and wash first with hot soapy water, then in plenty of running water followed by several rinses in distilled H,O Place 3 0 g gelatin m a beaker. Add enough boiling water to dissolve gelatin. Add water to total 600 mL. Add 0.3 g chromium potassium sulfate. Diethylpyrocarbonate (0.02%) can be added to the solution as an added precaution against RNases. DIP slides m the gelatin solution three times, then dry slides in a hot room overnight at 37°C

2.3. In Situ Hybridization 1. Phosphate-buffered salme (PBS). 2. 0.3% (v/v) Triton X-100 in PBS. 3 Proteinase K solution: 10 pg/mL in a solution of O.lMTris-HCl, pH 8.0,50 mM EDTA (stock proteinase K: 20 mg/mL, store at -20°C). 4. 4% paraformaldehyde m PBS (freshly prepared). 5. Acetylation solution: 0.25% (v/v) acetic anhydride, O.lM tiethanolamine, pH 8.0. Make fresh as required and use immediately. 6. 20X standard sodium citrate (SSC) stock: 3MNaCI,0,3Mtrisodium citrate Dilute as required. 7. Deionized formamide: It is deionized by mixing approx 5 mg mixed bed resm (Sigma, Poole, UK) per 50 mL formamide and stirring the mixture on a Magnestir for 30 min until the pH IS approx 7.0. The mixture IS then filtered and stored at -20°C. 8. IZNase A solution: 20 pg/mL in a solution of O.SMNaCl, 10 mA4Tris-HCl, pH 8.0, 1 mM EDTA (stock RNase A: 10 mg/mL, store at -20°C). 9. Hybridization solution: 50% formamide, 0.3MNaCl,20 mMTris-HCl, pH 7.5,5 n&f EDTA, 10 mA4 NaI-I$O,, pH 8.0, 10% dextran sulfate, 1X Denhardt’s solution and 500 pg/mL yeast tRNA This is prepared freshly from the following stock solutions, a. 100% deionized formamide. b 5MNaCI. c. 1M Tns-HCl, pH 7.5. d. 0.5M EDT.+ e. 1MNaH2P04, pH 8.0. f. 50% dextran sulfate. g. 50X Denhardt’s solution* 1% bovine serum albumin, 1% polyvinyl-pyrrohdone (PVP-360), 1% Ftcoll400. h. Yeast tRNA. Store solutions a, g, and h at -20°C and b-f at room temperature. 10. 70,90, and 100% ethanol containing 0 3M ammonium acetate.

In Situ Hybridization

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11. Autoradiographic emulsion: Ilford K-5 emulsion diluted 1: 1 with distilled Hz0 12. Kodak D-19 developer, Ilford stop bath, and Ilford fixer, prepared according to manufacturer’s instructions.

3. Methods (see Notes l-3) 3.1. In Vitro Transcription The following protocol is a modification of that given by Promega Biotec (Southampton, UK) for synthesis of RNA probes. 1. To a sterile microfttge tube, at room temperature, addin the followmg order: 4.0 & 5X transcription buffer, 2 pL 100 rnJ4 DTT, 1 uL RNasin, 4 pL nucleotide mixture, 1 pL 100 @fUTP,;l pL linearized plasmid template DNA (OS-l.0 pg), 6 pL [35S]UTPaS, and 1 pL SP6 RNA polymerase, T7 RNA polymerase, or T3 RNA polymerase to 20 pL final volume. 2. Incubate for l-2 h at 37°C. 3. To terminate transcription, add 1 pL of RNase-free DNase. Incubate at 37OC

for 15 min. 4. Add 3 pL yeast tRNA (10 mg/mL) as carrier. 5. Separate the probe from unmcorporated nucleotides using autoclaved sephadex

G-50 loaded in a sterile glasspipet. After pouring the colunm, run through 5 & of yeasttRNA (10 mg/rnL) and 1.O mL Tris-EDTA (TE) buffer + 10 mA4 DTT. 6. Load the sample and collect 200 JJL fractions by adding 200 pL aliquots of TE buffer + 10 mA4 DTT to the column. Remove 1 pL from each fraction for assessment of incorporation of radioactivity, count the radioactivity on a liquid scintillation counter, and from this determine the percent of incorporation of

radioactivity. 7. Pool fractions (usually three tubes), and add one-tenth vol 3M sodium acetate, pH 5.2 (0.25M final concentration), and 2.5 vol of absolute ethanol. Mix and leave at -70°C overnight. 8. Spin in a microfuge for 30 min. Discard the supematant. Dry the RNAJ pellet under vacuum. When dry, dissolve the pellet in 50 pL of 10 mA4 DTT. Remove 1 fi for counting of radioactivity. Use immediately or store3JSprobesat -7OOC. The maximum storage time will depend on the radioisotope used. However, background increases with storage time.

3.2. Tissue Preparation and Fixation of Sectioned Tissue Different fixatives are used in different laboratories. We recommend freshly made 4% paraformaldehyde in PBS, which gives the best results when considering both RNA retention and cellular morphology. 1. Tissues for hybridization must be collected as fresh as possible. Freeze the tissue block in a beaker containing isopentane that is chilled by hquid nitrogen. If one is to immediately use a tissue sample, it is often easier to mount the block directly onto a cryostat chuck before freezing. Store at -70% To avoid RNase contami-

nation, wear gloves anduse sterile equipment.

Mak and Barnes

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2. Cut sections (10-15 pm) on a cryostat at -2O’C (Bright cryostat, Huntingdon, UK) and thaw-mounted onto gelatin-coated glass slides and allow to dry before storing m a slide box with desiccant at -70°C. 3. Rinse the slides with tissue sections in cold PBS, and fix by immersion in 4% paraformaldehyde in PBS for 60 min at 4’C, before proceeding

3.3. In Situ’HybrkWafion Precautions should be taken to avoid RNase contamination tion is complete.

3.3.1. Prehybridization 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

until hybridiza-

Treatments of Tissue Sections

Rinse twice in PBS for 3 min. Permeabilize by immersion in 0.3% Triton X-100 in PBS for 15 min. Wash twice with PBS for 3 min. Deproteinize by incubation with 10 @nL proteinase K solution for 7.5 min at room temperature. Rinse in PBS for 3 min. Stop deproteinization by immersion in 4% paraformaldehyde in PBS for 5 min. Briefly rinse in DEPC-treated H,O (~30 s). Immerse in freshly prepared acetylation solution for 10 min to reduce nonspecific binding. Dehydrate in 70, 95, and 2X 100% ethanol (2 min each). Air dry before processing for hybridization.

3.3.2. Hybridization 1. After preheating to 80°C for 2 min to denature any RNA secondary structure, apply 20 pL of hybridization mixture containing 500,000 cpm/& of radiolabeled RNA probe diluted in hybridization solution over the tissue sections. 2. Incubate at 50-52’C overnight in a humid chamber (containing paper towels soaked in 50% formamide/2X SSC). Seal chamber with tape to prevent evaporation.

3.3.3. Posthybridization

Washing

1. Briefly rinse the slides with 4X SSC/lO mA4 DTT at room temperature. 2. Wash the slides with 4X SSC (37’C, 2 x 30 min) with gentle shaking. 3. Remove unhybridized single-stranded RNA probe by treating preparations with RNase A solution for 30 min at 37OC. 4. Wash the slides with 2X SSC (42’C, 30 min), with gentle shaking. 5. Wash the slides with 0.1X SSC (50°C, 30 min), with gentle shaking. 6. Dehydrate in 70,90, and 2X 100% ethanol containing 0.3M ammonium acetate (2 min each at room temperature). 7. Air dry (60-90 min).

In Situ Hybridization

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3.3.4. Autoradiography 1. Dip the slides in Ilford K-5 emulsion at 42’C in the dark room. 2. Air dry vertically in a rack for 1-2 h. 3. Place slides in light-tight black plastic boxes, each containing desiccant, and store at 4°C for 7-2 1 d, depending on the radioisotopes used, as well as the amount of radioactivity, which can be quite low in instances in which there are few mRNA copies/cell. Warm up slides to room temperature before development. 4. Develop in Kodak D-19 developer for 4 min. Briefly rinse in Ilford stop bath for 30 s. Fix in Ilford fixer for 5 min. All solution should be 20°C. 5. Wash slides in distilled water for 30 min. 6. Wash in cold, running tap water for 60 min.

3.3.5. Countersraining 1. Preparations are usually lightly counterstained with hematoxylin. 2. Dehydrate, clear, and mount with DPX. 3. View under light and dark field illumination.

4. Notes 1. There is no single control that ensures the labeling is specific in all respects, Therefore, control experiments are very important to assessthe specificity of the hybridization and should include the following: a. Sense probes: Probes identical to the coding strand of the mRNA under investigation are transcribed and hybridized as antisense probes. b. Ribonuclease treatment: Sections are pretreated with RNase A (20-100 pg/mL, 37Y!, 30 min) before hybridization. This removes all RNA from tissue and is not a test specific only to the message of interest. There is also the potential that signal will be removed by residual amounts of applied RNase that degrades subsequently applied RNA probes, and thus invalidates the results. c. Northern blot analysis: This yields a tissue mRNA signal of the appropriate size detectable with the same probe sequence as used for ISH. d. Test the extent of hybridization to an inappropriate probe for the tissue in question. e. Include samples of other tissues that are thought not to contain the message. f. When possible, examine a positive control: Use tissue that contains an abundant amount of the message of interest. g. Use several probes complementary to different regions of the same gene. h. Perform receptor autoradiographic mapping on adjacent sections to determine that cells that contain the receptor protein also manufacture the message. This will not always be the case (14-I 7). 2. Signal/background ratio: There is no single set of conditions for hybridization, or stringency of washing of tissue sections that will be suitable for all experiments. In general, the hybridization of a nucleic acid probe to a target nucleic acid is a reversible process; the strength of the duplex formed depends on the length of the

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probe, temperature, salt concentration, base composition, number of mismatches, and the concentrations of destabilizing agents such as formamide. The vanation in salt concentration, temperature, and formamide concentrations are critical in determinmg the stringency of hybndization or washing conditions. The stringency can be varied by raising or lowering the percentage of formamide and the temperature, thereby influencing the stability of duplexes on the tissue section. High nonspecific background binding of the probe might suggest that a more stringent hybrtdizatton is required. These conditions need to be determined empirically for each probe. After hybridization, sections are washed several times in salt solutions with the aim of retaining the maximum amount of specifically bound probe while washing off nonspecifically bound probe Washing, like hybridization, depends on temperature (the higher, the more strmgent) and salt concentration (the lower, the more stringent). The balance between these has to be determined empirically for each probe. Although probes labeled with 35Sgive better subcellular resolution than those labeled with 32P, there is an increase m background The background may be reduced by the following additions/modifications: decrease m autoradtographtc time; minimizatron of the amount of probe used for hybridtzation; the inclusion of dithiothreitol (DTT, 10 mA4) in hybndizatron mixture and posthybridization washings to prevent oxidation of thiol groups. 3. In vitro transcriptton: The maJor problem in working with mRNA preparations is RNase contammation. RNases are remarkably stable enzymes, surviving heating to lOO”C, so considerable care is required to ensure that all glassware and solutions are RNase free. RNases are also present on fingers, so gloves should be worn throughout the transcription and hybridization protocols. Glassware should be baked at 200°C for 4 h. All solutions should be prepared with DEPC-treated water. Diethylpyrocarbonate (DEPC, 0 l%, final concentration) is added to distilled water and left at 37’C for 12 h. Residual DEPC is destroyed by autoclaving the water. The transcription reaction can be run in the absence of unlabeled uridine triphosphate. For 20 pL reaction, 100 pC!i of >I000 Ci/mtnol [35S]UTP~S IS 5 @4 However, the yield of full-length transcripts drops as the concentratton of hmitmg nucleotrde undine triphosphate falls below 5 p,~I4,The ideal probe size is <300 bp, longer transcripts can be hydrolyzed m hydrolysis buffer (40 mM NaHC03, 60 mMNa2C03) heating to 60°C for various time, followed by neutralizing buffer (3214sodium acetate, pH 5.2). The integrity of the labeled probe can be verified by electrophoresis on denaturing agarose gels.

References 1. Gall, J. and Pardue, M. (1969) Formatron and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl. Acad. Sci. USA 63,378-383. 2. John, H. A., Birnstlel, M. L., and Jones, K. W. (1969) RNA-DNA hybrids at the cytological level. Nature 223,582-587.

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3. Buongionrno-Nardelh, S. and Amaldi, F. (1970) Autoradiographic detection of molecular hybrids between RNA and DNA in tissue sections. Nature 225,946-948. 4. Coghlan, J. P., Aldred, P., Haralambidis, J., Niall, H. D., Penschow, J D., and Tregear, G. W. (1985) Hybridization histochemistry. Anal Bzochem 149, l-28. 5. Penschow, J. D., Haralambidis, J., Darling, P. E , Darby, I A., Wintour, E. M., Tregear, G. W., and Coghlan, J. P. (1987) Hybridization histochemistry. Expenentia 43,741-750. 6. Varndell, I. M., Polak, J. M., Sikri, K. L , Minth, C. D., Bloom, S. R., and Dixon, J. E. (1984) Visualization of messenger RNA directing peptide synthesis by zn situ hybridization using a novel single-stranded cDNA probe. Hzstochemistry 81, 597601

7 Cox, K H., De Leon, D. V , Angerer, L. M., and Angerer, R. C. (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Blol 101,485-502. 8. Hoefler, H., Childers, H., Montminy, M. R., Lechan, R. M., Goodman, R. H., and Wolfe, H. J. (1986) In sztu hybridization methods for the detection of somatostatin mRNA in tissue sections using antisera RNA probes. Histochem J 18,597-604. 9. Young III, W. S. (1992) In situ hybridization with oligodeoxyribonucleotide probes, in In Situ Hybndlzation: A Practical Approach (Wilkmson, D. G., ed.), IRL, Oxford, pp. 33-44. 10. Guitteny, A. F., Fouque, B., Mougin, C., Teoule, R., and Bloch, B. (1988) Histological detection of messenger RNAs with biotinylated synthetic oligonucleotide probes J Hlstochem. Cytochem. 36,563-671. 11. McNicol, A., Farquharson, M., and’walker, E. (1991) Non-isotopic in situ hybridization with digoxigenm and alkaline phosphatase labelled ohgodeoxynucleotide probes. Applications in pituitary gland. Path01 Res Pratt. 187, 556-558. 12. Durrant, I., Brunning, S., Eccleston, L., Chadwick, P., and Cunningham, M. (1995) Fluorescem as a label for non-radioactive in situ hybridization. Histochem. J. 27,94-99. 13. Evans, M. R. and Read, C. A. (1992) 32P, 33P and 35S* selectmg a label for nucleic acid analysis. Nature 358, 520,521. 14. Hamid, Q. A , Mak, J C. W , Sheppard, M. N., Corrm, B., Venter, J C., and Barnes, P. J. (1991) Localization of beta2-adrenoceptor messenger RNA m human and rat lung using zn sztu hybridization: correlation with receptor autoradiography. Eur. J Pharmacol (Mol. Pharmacol. Section), 206, 133-138. 15. Mak, J. C. W., Baranmk, J. N , and Barnes, P J (1992) Localization of muscarinic receptor subtype messenger RNAs in human lung. Am J. Respw Cell Mol. Biol 7,344-348 16. Mak, J C. W., Haddad, E.-B., Buckley, N. J., and Barnes, P. J. (1993) Visualization of muscarinic m4 mRNA and M4 receptor subtype in rabbit lung Life Sci. 53, 1501-1508. 17. Mak, J. C. W., Nishikawa, M,, Haddad, E.-B., Kwon, O.-J., Hirst, S. J , Twort, C. H. C., and Barnes, P. J. (1996) Localization and expression of P-adrenoceptor subtype mRNAs m human lung. Eur J. Pharmacol , in press.

7 lmmunocytochemical Methods for Investigating Receptor Localization Maria I. Fonseca and R. Dale Brown 1. Introduction Immunocytochemical procedures offer a unique means to visualize receptors at cellular and subcellular resolution. This approach has been enhanced by the availability of immunochemical probes to simultaneously visualize cellular ultrastructure, such as intracellular organelles, cytoskeleton, proteins involved in signal transduction, and markers of cellular differentiation. The application of immunocytochemical methods, in conjunction with experimental manipulation of cellular function, has provided powerful Insights not only into the structural aspects of receptor localization and topology, but also into dynamic processes of receptor biology, including coupling to effecters, intracellular trafficking, ontogenesls, and disease. Numerous methodologies and protocols have been used for receptor immunolocalization at the light and electron microscopic level. These adaptations have been optimized for specific applications, but ultimately derive from basic immunochemical principles. This review will emphasize studies at the light microscopy level. We will briefly mention some general considerations that should be made before choosing the approach suited to the system at hand and the specific goals of the study, More detailed information may be obtained from several authoritative books (1-3).

7.1. Source of lmmunogen SuccessfU immunolocalization requires antibodies with sufficient affinity and specificity to recognize receptors in the native conformation, and at low abundance. This low abundance, inherent to most receptors m physiological systems,imposes the additional technical obstacle of obtaining sufficient quanFrom. Methods m Molecular Biology, vol 83 Receptor Sfgnal Transduction Edited by R A J Chalks Humana Press Inc , Totowa, NJ

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Protocols

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tities of antigen for immunization. Thus, the initial preparation of antibodies to the purified nicotinic acetylcholine receptor by Lindstrom and colleagues (4) depended on the availability of Torpedo electric organ, in which this receptor is highly enriched. Subsequent advances in protein sequencing, molecular cloning, and expression technologies have greatly facilitated the production of antireceptor antibodies by circumventing the need for quantities of purified receptor, and by allowing the design of antibodies directed against specific protein epitopes. The realization that immunogens dertved from bacterial fuston proteins (5,6) or synthetic oligopeptides (7,s) could be used to generate antibodies recognizmg native receptors provided sources of antigen independent of receptor purification. The advent of computer-assisted algorithms to predict sequence antigemcity, combined with increasing knowledge of the primary sequences and topologies for families of homologous receptors, makes it possible to optimize the production of specrfic antibodies by targeting eprtopes of known topology (e.g., intracellular vs extracellular domains) and favorable antigemcity. Strategtes for producmg antueceptor antibodies are covered in additional detail in Chapter 5. Finally, the procedure known as epitope tagging allows introduction of an exogenous peptide epttope of defined anttgenictty into the receptor primary structure by use of recombinant DNA technology. This approach provides a powerful alternative to more traditional antireceptor antrbodies when the experimental objectives are compatible with heterologous receptor expression (9).

1.2. Type of Antibody Both monoclonal and polyclonal antibodies (MAbs and PAbs) have been used successfully for receptor distribution studies. Since MAbs constst of a single antibody species that recognizes an individual antigenic epitope, they generally offer high spectticity and low nonspecific immunostaining. These properties make MAbs well-suited for electron microscopy and for studies in living cells. The generation of immortahzed hybridoma cell lines offers a potentially unlimited source of antibody. However, MAbs possessing low avidttles (a measure of the composite interaction affimties between antrgemc epitopes and antigen-binding sites m a multivalent antibody population) may give low signal or may not be sufficiently sensitive to detect low-abundance antigens such as receptors. Moreover, MAb production demands a stgnificant commitment of technical expertise, supporting resources, and effort to realize the goal of antibody production. MAbs can be used for immunostaming as hybridoma supematants, ascites fluid, or purified antibody preparations. PAbs contain heterogeneous populations of antibody species directed against different epitopes, which can result in high avidity for the antigen, Polyclonals

Receptor lmmunolocalization

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give generally good signal strength, but may suffer correspondingly from higher background immunoreactivities. This nonspecific staining can be reduced by purifying the IgG fraction on protein A- or protein G-Sepharose columns to remove serum contaminants. Immunoaffinity purification will also lower the background, since it will eliminate irrelevant contaminating antibodies. However, this procedure may paradoxically reduce antibody activity and staining sensitivity, since those antibody species possessing the highest antigen affinities may be incompletely recovered from the affinity matrix during purification. In our experience, it is advisable to observe the pattern of immunostaining obtained with crude antisera before proceeding to more highly purified preparations. 1.3. Direct vs indirect lmmunolabeling

Techniques

Direct immunolabeling procedures employ primary antibodies that themselves have been modified to allow visualization. The specificity of immunolocalization relative to nonspecific signal is good, since the same antibody. molecule confers both antigen binding and detection. However, direct immunolabeling lacks the amplification achieved with indirect labeling techniques, so that signal may be lower. Further, covalent modification of the antibody with the detection moiety can affect the antibody’s ability to recognize antigen. This limitation can be more serious for PAbs that interact with multiple sites on the target antigen. It is also necessary to purify the modified antibody to remove excess reagents that may interfere with mununodetection. Direct immunolabeling with MAbs avoids these ambigunies because of their biochemical homogeneity. Indirect labeling techniques employ unmodified primary antibodies that are visualized by subsequent binding with labeled secondary antibodies or antibody-binding proteins. The most common secondary detection method utilizes labeled antibodies prepared in an exogenous animal species against IgG from the animal species used for primary antibody production. (For example, goat antirabbit IgG are used as secondary antibodies to detect rabbit IgG primary antibodies.) The primary antibody also can be detected by labeled protein A or protein G, bacterial proteins that bind to Fc regions of IgG from several animal species. Additional variations to this procedure are the use of biotinylated secondary antibody followed by a labeled avidin-biotin complex, or the use of unlabeled secondary antibody followed by a peroxidase-antiperoxidase (PAP) complex. Indirect labeling procedures can detect very low antigen concentrations, since multiple secondary detection events occur for each primary antibody binding. As might be expected, nonspecific immunolabeling may also be elevated compared to direct immunolabeling protocols.

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1.4. Detection Methods Three principle methods of immunocytochemical detection have been utilized (reviewed in refs, 2, 3, and 10). Fluorescent labels are widely used, since they provide high sensitivity and spatial resolution to subcellular levels. They are unique in preserving the viability of living cells during labeling for real time measurements. These reagents are excellent for colocahzation procedures using multiple fluorophore labels with distinct emission spectra. However, they require more specialized mstrumentation for fluorescence microscopy, they are subject to photobleaching during illumination or storage of specimens, and they are not suitable for electron microscopic detection, Enzymatic labels, in contrast, provide lower spatial resolution, but greater sensitivity and stability, compared to fluorescence detection. Enzyme-conjugated antibody labeling has been widely used to map the distribution of receptors in tissues (notably, the brain) under the light microscope. Enzyme labeling techniques have also been applied to electron microscopy, although their lower spatial resolution limits their usefulness. Gold conjugates were originally developed for electron microscopy, in which they have become the reagents of choice because of their excellent spatial resolution. The availability of gold conjugates of distinct particle sizes allows double labeling procedures for colocalization experiments. More recently, gold labeling methodology has been modified for light microscopy. In the following sections, we describe detailed procedures that we have used for immunolocalization of ai-adrenergic receptors in cultured human embryonal kidney (HEK) 293 cells transfected and selected for stable expression of ala-receptor cDNA. These approaches were used to study agonist regulation of a,n-adrenergic receptor subcellular distribution and trafficking (II). For this purpose, we prepared a rabbit PAb directed against the carboxy terminal decapeptide derived from the cloned or a-adrenergic receptor cDNA (sequence: [Cl-SNMPLAPGHF). This sequence was initially selected by analyzing the primary sequences of a,-adrenergic receptor subtypes using PC-based software to identify decapeptides of unique sequence with favorable predicted antigenicities (Seqaid; ref. 12). Antibodies were characterized for specific nnmunoreactivity toward the ora- receptor by immunoblotting and immunocytochemical protocols. Receptor localization was performed by indirect immunofluorescence using both conventional and confocal microscopy. Colocalization experiments were performed by double labeling immunofluorescence. We have utilized these same protocols to immunolocalize alB-adrenergic receptors in cultured cells expressing endogenous receptors at densities comparable to those found in physiological systems (100-500 fmol/mg membrane protein). Modifications of these procedures also can be applied to

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immunolocalize receptors in tissue sections, and to detect receptors by enzymatic labeling. 2. Materials 1. HEK cells transfected and selected for stable expression of ora-adrenergtc receptors, cultured under standard adherent condmons on glass cover slips (grade #l) coated with poly-o-lysine (Sigma, St. Louis, MO). Wild-type HEK cells, which lack endogenous al-receptors, are used as negative control. 2. Phosphate-buffered salme (PBS): 140 mMNaC1, 3 mM KCl, 8 mA4 Na2HP04, 2 mMKH2P04, pH 7.4 3. Fixative solution: PBS containing 4% formaldehyde. 4 Blocking solution: PBS containing 1% bovine serum albumin (BSA), 10% normal goat serum (NGS), and 0.01-0.05% Triton-X100. 5. Primary antibody for am-adrenergic receptor: rabbit polyclonal antlpeptide antibody specific for am-adrenergic receptor. 6. Primary antibody for transferrin receptor: purified mouse monoclonal antitransferrm receptor antibody for use as an endosomal marker (Amersham Life Sciences [Arlington Heights, IL] or Boehringer-Mannheim [Indianapolis, IN]), 7. Secondary antibody for ala-receptor localization: goat antirabbit IgG (H+L), FITC conjugate, preadsorbed with human, mouse, and rat serum proteins (Jackson Immunoresearch, West Grove, PA). 8. Secondary antibody for transferrin receptor localization: goat antimouse IgG (H+L), lissamine rhodamme conjugate, preadsorbed with human, mouse, and rat serum proteins (Jackson Immunoresearch). 9. Slow Fade mountmg medium (Molecular Probes, Eugene, OR).

3. Methods 3.1. Preparation

of Cells for Labeling

1. Cells are seeded into 35-m tissue culture dishes containmg sterile glass coverslips coated with poly-o-lysine according to the manufacturer’s instructions, and grown 2-3 d to subconfluent density, allowing good microscopic resolution. 2. Growth medium IS removed by aspiration and the cells are washed twice for 5 mm each with aliquots of PBS.

3.2. Fixation 1. Fixative solution is added to the cells and incubated at room temperature (RT) for 15 min (see Note 1). 2. Cells are washed three times for 5 min each wrth aliquots of PBS.

3.3. Permeabilization and Blocking Fixed cells are incubated with blocking solution for 2 h at RT (see Notes 2 and 3).

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3.4. Primary Antibody Reaction (Single or Double lmmunolabeling) 3.4.1. Single Antibody Labeling Blocking solution is removed by aspiration and primary antireceptor antibody diluted in fresh blocking solution is added (see Notes 4-6). Incubation proceeds at 4°C overnight (see Note 7). Cover slips can be left in the original culture dish for this step, but a larger volume of antibody 1s needed. If antibody is limited, cover slips can be removed, placed specimen-side down on a 50- to 100~$ drop of antibody solution applied to a layer of parafilm, and sealed inside a Petri dish containing water-saturated filter paper or cotton balls, to maintain humidity.

3.4.2. Double Antibody Labeling For colocalization experiments to demonstrate intemahation of orn adrenergic receptors to endosomes, a MAb against transferrin receptor is used m combination with the antibody directed against orn-receptor. Following removal of the blocking solution in Section 3.3., antitransferrin receptor antibody diluted 1: 100 in a fresh aliquot of blocking solution (per manufacturer’s recommendation) is added to the specimen for 3 h at RT. A concentrated aliquot of anti-o in-receptor antibody is then added directly into this incubation solution to give the final desired concentration, and the incubation is continued as described above for single immunolabeling. 1. Incubation with the indicated primary antibodies in Sections 3.4.1. and 3.4.2. is terminated by washing the specimen with three ahquots of PBS for 5 mm each (see Note 8).

3.5. Secondary Antibody Reaction (Immunofluorescence Detection) 1. Cells are incubated next with appropriate secondary antibodies, whrch are diluted 1:400 in blocking solution, for 1 h at room temperature (per manufacturer’s specifications). cxrB-Adrenergic receptor is visuahzed with FITC-conjugated goat antirabbit secondary antibody, and transferrin receptor is visualized with lissamine rhodamine conjugated goat antimouse secondary antibody. In dual labeling experiments, both secondary antibodies are diluted mto the same aliquot of blocking solution for incubation with the sample (see Notes 9 and 10). 2. Unbound secondary antibodies are removed by three 5 mm washes m PBS. 3. Cover slips are mounted using Slow Fade mounting solution (see Note 11) as follows. It is it imperative that the specimen not go dry during this process. The final PBS wash is aspirated and the specimen is immediately covered with a drop of mounting solution The cover slip is inverted onto a glass microscope slide, and the edges are sealed from air with nail polish.

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4 Cells are observed under a conventional epifluorescence microscope. Photographs are taken using Kodak Tn-X PAN 400 ASA film (see Note 12). The same specimens are also suitable for visualization under the confocal laser microscope in order to obtain higher spatial resolution of receptor localization (see Note 13). Scanning of l-p-thick sequential optical sections of individual cells is performed on single- or double-labeled specimens.

3.6. Alternative Procedures The procedures described here for immunofluorescence detection of receptors in cultured cells can be adapted for tissue sections (see Note 14), and for enzymatic detection methods (see Note 15).

4. Notes 1. The use of aldehyde fixatives to crosslink cellular protems IS a common procedure, since it preserves cell morphology and is mild enough to maintain unmunoreactivity of antigenic epitopes. Fixative solution should be prepared fresh by dilution of commercially available 37% formaldehyde solution (Sigma) in PBS. Alternatively, paraformaldehyde solution (4% w/v, prepared from the dry chemical) can be utilized as fixative. Fixation can also be performed with organic solvents such as acetone, methanol, or ethanol, or combinations of these; however, cellular morphology is not as well preserved as with crosslmkmg fixatives. Organic solvent fixation can be performed at room temperature or at -20°C (3). 2. Blocking and antibody solutions routinely use PBS or Tris buffered saline (TBS: 25 mM Tris-HCl, 140 mM NaCl, 3 mM KC 1, pH 7 4) as diluent. The addition of high concentrations of proteins into the blocking solution is used to minimize nonspecific binding of antibodies to the specimen. Some of the more commonly used protein blockers are BSA (l-3%), nonfat dry milk (5-lo%), and gelatin (0.3-l%). The addition of nonimmune serum (l-10%) from the same species as the labeling antibody (i.e., normal goat serum in the present example) to the blocking solution also contributes to lower backgrounds by reducing interactions of antibodies with immunoglobulin-binding sites in the tissue. In some protocols, proteins and nonimmune sera are added only during the initial blocking step, but omitted during prrmary and secondary antibody incubations, because of potential interference with antibody labeling. In our hands, the attendant increase in background signal lessens the benefit of this modification. 3. Permeabilization of the cell with detergents is necessary, particularly when using aldehyde fixation in order to allow antibody access to intracellular epitopes. Organic solvent fixation, by contrast, renders the membrane permeable without detergent addition. The more commonly used detergents and thetr effective concentrations are Triton X-100 (0.1-0.5%) and Nonidet P-40 (now marketed as Igepal CA-630, 0.2%). The choice of detergent and concentrations used will depend on the experimental system and should be optimized by the user to allow penetration of the antibody without compromrsing cell structure or antigemcity.

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4. Care m storage and handling of antibody solutions produces direct benefits in the strength of the immunostaining signal, and in maintaining low background reactivity. Concentrated stock solutions of primary and secondary antibodies or antisera should be dispensed into small aliquots and stored at -20 or -70°C in order to avoid repeated freezing and thawing cycles that can cause aggregation of antibodies and loss of activity. It IS not recommended that aliquots be thawed more than once Concentrated antibody solutions can be stored at 4°C for limited times (weeks to months). However, a preservative should be added to prevent bacterial growth. The most commonly used is sodium aside (0.02%). The user should be aware NaN, is toxic and interferes in many biological assays, particularly peroxidase immunodetection. Antibodies can also be sterilized by filtration. Crude antisera should be centrifuged 15-30 min at 15,OOOgbefore dilution mto blocking solution. Working dilutions of antibodies in blocking solution are preferably prepared fresh, or stored at 4Y! no more than l-2 d before use. 5. Antibody concentrations that yield the highest specific signal relative to the lowest background must be determined experimentally. The optimum antibody concentration will be determined by the avidity of the antiserum and the concentration of antigen in the sample For purified MAbs or affinity-purified PAbs in which the specific antibody concentrations are known, concentrations of 0.1-10 pg/mL should be tested. When the concentration of the antibody species of interest is unknown, serial dilutions ranging from 1: 1O-1 : 10,000 should be tested. 6. Immunocytochemical detection is based on the binding interaction between antigen and antibody. In common with other binding measurements in biological systems, the total observed immunostaining reflects both specific and nonspecific components, particularly when PAbs are employed. A key component of establishing the specific immunostaining signal is the use of appropriate controls. Particular caution should be exercised when investigating a novel antibody in order to demonstrate self-consistent immunostaining behavior toward complementary control and experimental conditions. Selection of controls will depend on the type of antibody and the nature of the antigen, as follows. An important control for a PAb is the preimmune antibody fraction collected from the same animal prior to immunization with the antigen. Normal (nonimmune) serum pooled from the same animal species may be substituted if the preimmune control is unavailable. Preimmune controls should be matched to the same concentrations (or dilutions) and equivalent purity as the test antibody. Thus, preimmune serum should be compared to antireceptor serum, and protein A-purified preimmune IgG should be compared to antireceptor IgG. A control fraction for affinity-purified antibodies can be prepared by adsorbing preimmune serum to the immobilized antigen used for affinity purification, and isolating the corresponding antibody eluate. This fraction will contain any components that nonspecifically bind to the affinity resin or that crossreact with the antigen. Comparison of affinity-purified antibodies with the purified preimmune IgG fraction is also recommended. The investigator should be aware that exposure of animals

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to extraneous antigens during the course of immunization can produce spurious immunoreactivities, so that preimmune controls alone may not be sufficient to establish antibody specificity. Useful controls for MAbs are the culture supernatants from control hybridoma cell lines (commercially available from American Type Culture Collection [Rockville, MD]), ascites fluid obtained from animals infected with unrelated hybridomas, or the corresponding purified antibody fractions. Test and control antibodies should be matched to the same immunoglobulin subtypes. An important control experiment for antibodies directed against synthetic peptides is to block the specific immunoreaction by preadsorption of the antibody with the cognate synthetic peptide. This experiment can be performed on antibodies at any stage of purity. A concentrated stock solution of peptide is added at a final concentration of 1e-1 W3A4into the desired working concentration of the primary antibody in blocking solution. Excess peptide concentration should be used to block all antigen-combining sites in the population, and will vary accordmg to titer and avidity of the antibody. The mixture is Incubated with gentle agitation overnight at 4’C, then used the following day in the immunolabeling protocol, as described in Section 3.4. An aliquot of nonadsorbed primary antibody should be processed through the same incubations and used to immunostam a companion specimen to demonstrate positive reactivity. Successful peptide adsorption can provide a convincing demonstration of antibody specificity, but it may not be effective in all cases. For example, solution-phase peptide may compete poorly if the antigen in the specimen is present in locally high concentration, or in a conformation that interacts favorably with the antibody. An alternate strategy in this case is to perform preadsorption with peptide nnmobilized on a matrix, and then use the antibody-depleted supernatant for immunostaining. Finally, additional experiments can test whether the behavior of the immunostaining stgnal is consistent with known features of receptor biology. Thus, intracellular epitopes should require permeabilization of the cell to detect immunoreactivity, and extracellular epitopes should be detectable without permeabilization (8). Cells or tissues that do not express the receptor of interest provide a further negative control. This experiment is most straightforward when working with heterologous receptor expression, in which wild-type and transfected cells can be compared directly. Some of these considerations are illustrated in Fig. 1. Figure 1 (A,B) shows immunostaining of HER/am transfectants with protein A-purified antireceptor IgG and the correspondmg protein A-purified preimmune IgG fractions, respectively. Positively stained cells exhibit significantly brighter immunofluorescence, which appears to distribute uniformly over the cell surface. Cell profiles show characteristic rim staining around the cell periphery, as would be expected for receptor localized to the plasma membrane. By contrast, the negative controls show much lower overall mnnunofluorescence. No rim staining is observed, leaving a diffuse background staining that cannot be localized with defined cellular structure in any focal plane. Crude antireceptor serum produces a pattern of

Fonseca and Brown

Fig. 1. Demonstration of immunostaining specificity of al,-adrenergic receptor antibodies in HEK cells. Immunostaining procedures were performed as described in Methods. All experiments used pooled populations of HEK/a,n transfected cells, except as noted. Immunostaining was performed with protein A-purified IgG fractions, used at 1: 1000 dilution. Images were obtained from replicate specimens in the same experiment and processed identically for immunostaining and microphotography. (A) Immunostaining with protein A-purified antireceptor IgG. (B) Preimmune IgG control. (C) Preadsorption with cognate peptide (1 W3M). (I)) Immunostaining in wild-type HEK cells. (original magnification x 156.)

immunostaining that is very similar to purified antireceptor IgG (data not shown). However, background staining with preimmune serum appears more elevated and nonuniform compared to the preimmune IgG fraction. For these reasons, the purified antireceptor IgG fraction is judged to give a cleaner signal, and is utilized in subsequent experiments. Figure 1C demonstrates the blockade of specific immunostaining by preadsorption of antireceptor IgG with the cognate receptor peptide sequence. Figure 1D further shows that immunoreactivity is not observed in wild-type HEK cells. Finally, omission of Triton Xl00 from the incubation buffers eliminates the positive immunostaining signal, consistent with

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Fig. 2. Agonist-induced redistribution of a,,-adrenergic receptors in HEK/atn transfectants. Cells were incubated for 1 h in physiological buffer containing no further additions (A), or 10 $4 norepinephrine (B), and then processed for receptor immunocytochemistry using protein A-purified antireceptor IgG as described in Methods. (original magnification x125) (Reprinted with permission from ref. II.) the predicted intracellular location of this carboxy-terminal epitope (data not shown). These results are extended in Fig. 2 (A,B), which shows control HER/ am-cells incubated in physiological buffer, and a companion culture treated with buffer containing the agonist norepinephrine. A clear redistribution of aiu-receptor occurs in the agonist-treated cells, so that nonuniform aggregates of immunofluorescence now appear within the cell and associated with the cell periphery. Taken together, the control experiments in Fig. 1 provide the investigator with a clear sense of the nature of specific vs nonspecific immunostaining in the preparation at hand. This observational base forms an essential foundation for subsequent mechanistic studies of receptor biology, as shown in Fig. 2. Because signal strength in immunocytochemical experiments is commonly evaluated qualitatively rather than quantitatively, successful interpretation of immunostaining results depends critically on the observational skill of the investigator, and on a systematic body of control experiments, such as these. 7. Times and temperatures of primary antibody incubations should be adjusted experimentally for optimal immunoreaction. Shorter incubations (l-3 h) are performed at room temperature or 37°C. Humidified chambers can be used to avoid evaporation when working with small antibody volumes. Bacterial overgrowth at elevated reaction temperature should be prevented by working under sterile conditions. More prolonged antibody incubations (16-48 h at 4’C) allow increased antibody dilutions (lower antibody concentrations), which may lower backgrounds while allowing PAbs with heterogeneous avidities to react to completion. Inclusion of a 2-3 h room temperature equilibration following reaction at 4OC is sometimes helpful to allow desorption of more weakly bound nonspecific

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antibodies, while binding of high avidity antibodies is maintained. These protocols are less useful for MAbs, which possess homogeneous avidities. Care must be taken to insure the integrity of the specimen and the activity of the antibodies during prolonged incubations. 8. Increasing the number and length of washes can help to lower the background, again by selectively dissociating more weakly bound antibodies. The wash buffer can also be supplemented with detergents, such as Triton (0.2-0.5%), NP40 (O.l%), or Tween-20 (0.05-0.2%), to reduce background. It is important to momtor specimen morphology during extensive washing to prevent degradation. 9. Autofluorescence (that is, artifactual fluorescence produced by endogenous fluorophores, such as flavinoids, present in the specimen) can severely interfere with fluorescence immunodetection. Autofluorescence can be determined by examming unstained specimens, using the same excitation and emission filters as are used for fluorophore detection. Autofluorescence is most commonly encountered in tissue sections, working at excttatton wavelengths of 400-450 nm (violet-blue to blue light), so that interference with fluorescein detection can be a parocular problem. Autofluorescence drmimshes at longer spectral wavelengths, and selection of alternate fluorophores is recommended in this situation. 10. Double-labeling experiments utilizing indirect immunodetectron require that the reagents for secondary detection specifically recognize the appropriate primary antibody without crossreaction. In our example, this specificity is based on using primary antibodies raised in different host species. Secondary reagents should be checked explicitly for crossreactivity with the primary antibodies m control experiments. In addition, crossreactivities of the secondary antibodies toward the specimen should be determined by immunostaining in the absence of primary antibody. Fortunately for present day immunocytochemists, there is an increasing repertoire of commercially available, affinity-purified secondary antibodies conjugated with a variety of fluorophores and preabsorbed with immunoglobulins and serum proteins from other species, to avoid crossreactivities and to allow multiple-labeling protocols. When labeling multiple types of antigens in a single specimen, it is essential to select fluorophores for immunodetection whose emission spectra do not overlap to allow selective visualization with the standard filter sets available for fluorescence microscopy. Tetramethyl rhodamine (red emission) and fluorescein (blue-green) have been used extensively for this purpose. More recently, ltssamme-rhodamme and Texas Red have been substituted for tetramethyl rhodamine, because of their improved spectral separation from fluorescein; and aminomethyl coumarin (AMCA) has been substituted for fluorescein to provide improved spectral separation from rhodamine. However, it is still important to verify experimentally the absence of fluorescence bleed-through. Singly labeled specimens should show fluorescence only when viewed with the appropriate emission filter set, and not when viewed with filter sets correspondmg to other fluorophores.

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11. Certain fluorophores, notably fluorescein, are prone to photobleaching under prolonged tllumination or storage. The use of commercially available antioxidants, such as n-propyl gallate, in the mounting medium will reduce photobleaching. It is also advisable to limit the duration of exciting radiation. When feasible, the initial inspection and focus of specimens should be performed under phase-contrast illumination, or at lower intensity fluorescent illumination. Specimens should be stored at -20°C both to limit photobleaching and to preserve cellular morphology. In addition, we strongly recommend making the mitial investment of labor to make photographic records of specimens at the time of experimentation, rather than during the later stages of manuscript preparation and revision 12. Photography of mununofluorescence typically entails working at low light levels, unavoidable photobleaching during sample illumination, and the need to minimize photographic exposure times for maximum brightness and contrast over background. ASA 400 film is usually recommended, because it provides good sensitivity while preserving image definition. 13. The confocal microscope employs a focused illumination spot and detection pinhole, in concert with computer-assisted data acquisttion, to form an image from a discrete optical section wtthin the focal plane. Fluorescence outside this focal plane is eliminated, defining a depth of field as small as 0.2 pm. A considerable increase m contrast and resolution is achieved compared to conventional microscopy. Figure 3 illustrates the use of confocal microscopy to examme agomststimulated redistribution of a m-receptors in HEK cells. In this experiment, confocal images of ala-receptor immunofluorescence, obtained at comparable focal planes relative to the attachment surface, are shown for control (Fig. 3A) and agonut-treated cells (Fig. 3B). Immunostaining m control cells at this focal plane is localized to the cell periphery, consistent with the rim staining observed in Figs. 1 and 2. Agonist-treated cells show bright and nonuniform aggregates of immunofluorescence distributed internally, and associated wtth the cell surface. These images demonstrate that agonist elicits a dramatic internalization and redistributron of am-receptors in this system. The enhanced resolution obtained by this technique is evident by comparison of Fig. 3 with conventional epifluorescence images in Fig. 2. Confocal microscopy is the method of choice for subcellular localization of receptors, and for colocalizatton of receptors with other cellular markers. Serial optical sections can also be combined by computatton to reconstruct images m three dimensions. The reader should consult refs. 13-15 for a more complete description of this powerful methodology. 14. The procedures described here can be applied to mrmunofluorescence labeling of receptors in tissue sections. Detailed descriptions for preparation of tissue sections in receptor immunolocalization should be consulted (2,3,26). Briefly, the gentlest method for antigen preservation is to prepare tissue sections from frozen, unfixed tissues. Alternatively, tissue is prefixed by perfusion of the isolated organ or the whole animal, then cryoprotected, frozen, and sectioned. This approach works well for preserving tissue morphology. Sections prepared by

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Fig. 3. Agonist-induced orn -receptor redistribution observed with confocal microscopy. Cells were incubated for 3 h in physiological buffer containing no agonist addition (A), or 10 @4 norepinephrine (B), processed for receptor immunofluorescence, and visualized by confocal microscopy (optical section at z = 2 pm above attachment surface). (Scale bar = 10 pm.) (Reprinted with permission from ref. 11.) either method then are processed for immunostaining as described in the following paragraphs. Sections (usually 5-10 pm thickness) may be attached to gelatincoated slides prior to the immunostaining procedure, or alternatively, are

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processed free-floating to facilitate antibody penetration, and mounted onto slides at the end of the procedure. 15. Enzymatic immunodetection with horseradish peroxidase-conjugated reagents is another common procedure for immunolabeling receptors, particularly m tissue sections. The followmg modification of the protocol described for fluorescentlabeled secondary antibodies can be utilized (16; see also refs. 2, 3, and 10). Incubatton with the HRP-labeled secondary antibody (Section 3 5.) 1sperformed according to the conditrons specified by the manufacturer The specimen 1s then washed twice for 5 mm with PBS, followed by one wash for 5 min with ammomum phosphate buffer (APB* O.lMNaH,PO,, pH adjusted to 7.4 with NH,OH). The wash is removed and enzymatic activity is developed with APB containing 0 5 mg/mL diaminobenzidme tetrahydrochloride, 0.03% H,O,, and 0 04% NrCl, as enhancer. The developing solutron should be prepared immediately before use and the reagents added in the order given. Concentrated stock solutions of diaminobenzrdine and N&I, may be prepared individually m water and stored frozen m ahquots, if desired Dtaminobenzidine is carcmogenic, and the substrate should be handled and disposed with care. Incubation with the developing solution continues until a black color appears in the specimen (2-20 mm). It should be stopped when the colored reaction reaches its peak, by washing the specimen with APB followed by PBS The presence of sodmm azide m any of the buffers will interfere with the reaction The specimen 1s mounted in gelatin-glycerol mounting media (available commercrally). Specimens should be stored in the dark, preferably at -2O’C The signal obtained with the enzymatic label is quite stable. Specimens are observed and photographed under brightfield optics Nonspecific reaction resulting from endogenous peroxidase activity in the specimen should be determined by a control experiment omitting the secondary antibody. This background activity can be removed by treating the specimen with PBS containing 0 5-2% HzOz for 10 mm, followed by two 5-min washes with PBS prior to blockmg step (see Section 3.3.).

Acknowledgments This work was supported in part by NIH grant GM 41470 to RDB. The authors are grateful to Dr. Brenda Russell for use of fluorescence microscopy mstrumentation, and to Dr. Herbert Proudfit for critically reviewing the manuscript.

References 1. Sternberger, L. A. (1986) Immunocytochemzstry John Wiley, New York 2. Larsson, L.-I. (1988) Immunocytochemistry Theory and Practzce. CRC, Boca Raton, FL. 3. Harlow, E. and Lane, D. P. (1988) Antibodies- A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Sprmg Harbor, NY. 4. Lindstrom, J. B , Einarson, B., and Tzartos, S. (1981) Production and assay of antrbodies to acetylcholme receptors. Methods Enzymol. 74,432-460.

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5. Carroll, S B. and Laughon, A,, (1987) Production and purification of polyclonal antibodies to the foreign segment of P-galactosidase fusion proteins, in DNA Cloning: A Practical Approach, vol. 3 (Glover, D. M., ed ), IRL, Oxford/Washington, DC, pp. 89-l 11. 6. Wall, S. J., Yasuda, R. P., Hory, F., Flagg, S., Martin, B. M., Ginns, E. I., and Wolfe, B. B. (1991) Production of antisera selective for ml muscarininc receptors using fusion proteins: distribution of ml receptor in rat brain Mol. Pharmacol 39,643-649. 7. Doolittle, R. F. (1976) Of URFS and ORFS: A primer on how to analyze derived amino acid sequences. Umversity Science, Mill Valley, CA. 8. Wang H.-y., Lipfert, L., Malbon, C. C , and Bahouth, S. (1989) Site-directed antipeptide antibodies define the topography of the P-adrenergic receptor. J Bzol Chem 264, 14,424-14,43 1. 9. von Zastrow, M. and Kobllka, B. K. (1992) Ltgand regulated internalization and recycling of human &-adrenergic receptors between plasma membrane and endosomes containing transferrin receptors. J. Biol. Chem. 267,3530-3538. 10 Manson, M., ed. (1992) Immunochemical Protocols, in Methods in Molecular Biology, Chs. 10, 11, 15, 17. Humana, Totowa, NJ 11. Fonseca, M. I., Button, D. C., and Brown, R. D. (1995) Agonist regulation of a,,-adrenergic receptor subcellular distribution and function. J. Bzol Chem 270, 89024909. 12 Rhoads, D. D. and Roufa, D. J. (1985) Emetine resistance of Chinese hamster cells: structure of wild type and mutant nbosomal protein S 14 mRNAs. Mol. Cell Biol. 5, 1655-1659. 13. Wright, S. J., Centonze, V. E., Stricker, S. A., De Vries, P. J., Paddock, S. W., and Schatten, G. (1993) Introduction to confocal microscopy and three dimensional reconstruction, in Cell Biological Applications of Confocal Mxroscopy, vol. 38, Methods in Cell Biology (Matsumoto, B., ed.), Academtc, Orlando, FL, pp. l-45. 14. Clark Brelje, T., Wessendorf, M. W., and Sorenson, R. L. (1993) Multicolor laser scanning confocal immunofluorescence microscopy: Practical applications and limitations, in Cell Biological Appkcations of Confocal Mwroscopy, vol. 38, Methods in Cell Bzology (Matsumoto, B., ed.), Academic, Orlando, FL, pp. 97-l 8 1. 15. Shotton, D. M. (1989) Confocal scanning optical microscopy and its applicattons for biological specimens. J Cell SCL 94, 175-206. 16. Fonseca, M. I., Aguilar, J. S , Skorupa, A. F., and Klein, W. L. (1991) Cellular mapping of m2 muscarmic receptors m rat olfactory bulb using an antiserum raised against a cytoplasmic loop peptide. Brain Res. 563, 163-170.

8 Measurement of Agonist-Stimulated [35S]GTPyS Binding to Cell Membranes Sebastian

Lazareno

1. Introduction

This chapter describes a functional assay that measures the increase in guanine nucleotide exchange at G proteins in cell membranes, resulting from agonist binding to G protein-coupled receptors (GPCRs), by monitoring the binding of a radiolabeled, hydrolysis-resistant analog of GTP, [35S]GTPyS, in the presence of unlabeled GDP. The function of GPCR activation is to stimulate GTP/GDP exchange at G proteins (1) (Fig. 1). In a cell, the guanine nucleotide exchange cycle is initiated by the binding of an agonist-occupied (or “activated”) GPCR to a heterotrimeric G protein in the cell membrane. This stimulates the dissociation of GDP from the a-subunit of the G protein, allowing endogenous GTP to bind in its place. This in turn causes the dissociation of the receptor and the Go-GTP and Gj3y-subunits of the G protein. The Go-GTP and Gpy-subunits can each activate effecters, such as adenylyl cyclase, PLC, and ion channels (1). The Go-GTP is inactivated by an intrinsic GTPase, which hydrolyzes the GTP to GDP; Go-GDP in turn inactivates the GPy by binding to it, resulting in an inactrve GDP-containing heterotrimeric G protein ready for the next activation cycle. This process can be monitored in vitro by incubating cell membranes containing G proteins and GPCRs with GDP and [35S]GTPyS. Binding of [35S]GTPyS, like GTP, stimulates dissociation of Gcz-[~%]GTP~S and Gj3y, but, unlike GTP, [35S]GTPyS is relatively resistant to hydrolysis by the intrinsic GTPase of Go, dissociates slowly from Go, and therefore accumulates in the membranes. The effect of receptor activation on [35S]GTPySbinding in the presence of GDP is catalytic, i.e., it increases the rate of [35S]GTPyS binding From

Methods tn Molecular Bfology, vol 83 Receptor SIgnal Transduct/on Edlted by R A J Challiss Humana Press Inc , Totowa, NJ

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Pi -GDP \

+GTP

Fig. 1. Guanine nucleotide (GDP/GTP) exchange cycle at G proteins (c&y) stimulated by agonist (A) binding to receptor (high agonist-affinity state, R,, or low agonist-afftnity state, RL). rather than the equilibrium level of [35S]GTPyS binding. So this is intrinsically a nonequilibrium binding assay. The assay of agonist-stimulated [35S]GTPyS binding has a number of useful features: 1. It can use the same membrane preparations and assay conditions used in studies that measure radioligand binding to the GPCR, thus allowing a direct comparison of agonist action and agonist occupancy of the receptor; 2. It allows a measure of agonist action in systems in which the subsequent effector mechanisms are unknown; 3. It allows a direct comparison of receptor activation with receptors that activate different G protein regulated effector systems; and 4. It is convenient, easy, quick, and relatively accurate. The method used to measure agonist-stimulated [35S]GTPyS binding modification of methods described by Jakobs and colleagues (2,3).

is a

2. Materials 1. [35S]GTPyS (1000-1400 Ci/mmol) (see Note 1). 2. Cell membranes: Prepare as described in Section 3.1. 3. Homogenizing buffer: 20 mM HEPEWNa HEPES, pH 7.4, 10 mM EDTA. Protease inhibitors and reducing agents may also be included if required (see e.g.,

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Sweeney [4/), It is convenient to prepare or purchase stock solutions of 500 mM EDTA, which is stored at room temperature, and 1M HEPES/Na HEPES, which is stored at 4“C. A mixture of equal volumes of 1M HEPES and 1M Na HEPES should have a pH of about 7.5 when diluted to 20 m&f. 4. Membrane storage buffer: 20 mM HEPES/Na HEPES, pH 7.4,O. 1 mM EDTA. 5. Assay buffer: 20 mMHEPESMa HEPES,pH 7.4,lOO mA4NaC1, 10 mMMgC12, 1 l.& GDP (see Note 2). 6. Washing buffer: 10 mM sodmm phosphate buffer, pH 7.4 (see Note 5). A mixture of Na,HPO, and NaH,PO,, each at 10 n&f, in the ratio 1.4:0.6 v:v, has a pH of about 7.4.

3. Methods 3.1. Preparation

of Membranes

1. Wash the cells twice with 10 mL ice-cold homogenization buffer and scrape them off the plate with a teflon spatula and 2X 3 mL ice-cold homogenization buffer 2 Homogenize the cells with three or four 5-s bursts of a Polytron homogenizer (setting 6, with 30 s on ice between bursts). 3. Dilute the homogenate to 30 mL with ice-cold homogenization buffer and centrifuge at 40,OOOg for 10 min at 4°C. 4. Discard the supernatant and rehomogenize the pellet in 30 mL ice-cold membrane storage buffer. Centrifuge at 40,OOOg for 10 min at 4°C. 5. Repeat step 4. 6. Resuspend the pellet in about 5 mL ice-cold membrane storage buffer. 7. Determme the protein content (see Note 3). 8. Dilute the membrane preparation to 2 mg/mL protein with ice-cold membrane storage buffer and store in 0.5 mL aliquots at -7O’C.

3.2. p5S]GTPyS Binding Assay: Measurement

of Agonist Effect

1. Thaw an aliquot of frozen membranes and dilute to a concentration of 20 ~18protein/ml in ice-cold assay buffer containing 1 cln/iGDP (see Note 2). Store on ice. 2. Prepare 5-mL polystyrene test tubes in triplicate (see Note 4) with 10 clr, of each test agent made up to 100 times the final concentration in the assay 3. Thaw an aliquot of [35S]GTPyS and dilute a portion to a concentration of about 10 nM in assay buffer; if the original [35S]GTPyS has been stored at lo-fold dilution, then dilute it a further lOO-fold (see Note 1) 4. Add the diluted [35S]GTPyS to the diluted membranes, 10 pL label for each mL of membranes, and mix well. At 4”C, in the presence of GDP, binding of 0.1 ml4 [3sS]GTP# is very slow. 5. Distribute 1-mL aliquots of membranes + GDP + [3sS]GTPyS to the prepared tubes. 6. Incubate the samples at 30°C for 30 min. 7. Filter the samples over wetted (with water) glass fiber filters (Whatman GF/B) and wash twice with 3 mL ice-cold washing buffer (see Notes 5,6, and 13).

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8. Distribute the filter disks to scintillation vials and add scintillation fluid (see Note 7). 9. Distribute 10 pL diluted [35S]GTPyS (or 100 pL membranes + label) in duplicate to scintillation vials for measurement of added label. 10. Determine the radioactivity in each sample using scintillation spectroscopy (see Note 7).

3.3. p%]GTPyS Binding Assay: Measurement of Antagonist Affinity Potent antagonists often have slow dissociation kinetics, so expose the membranes to the agonist and antagonist for sufficient time, e.g., 30 min, for binding equilibrium of both ligands to be attained before addition of [35S]GTPyS to the assay. 1. 2. 3. 4. 5. 6. 7.

See Note 8 for experimental design. Perform steps l-3 of Section 3.2. Distribute 1-mL membranes + GDP to the prepared tubes. Incubate the samples at 30% for 30 mm. Add 10 pL [35S]GTPyS to the samples. Perform steps 6-10 of Section 3 2 See Note 9 for data analysis.

3.4. t%]GTPyS Binding Assay: Quantitation of Allosteric Modulator Some GPCRs, such as the muscarinic and adenosine A1 receptors, contain a second site at which agents can bind, with the effect of modulating the affinity of a directly acting ligand (such as the endogenous agonist) (5,. 1. Use a Schild analysis design (see Note 8). 2. If the allosteric agent has rapid dissociation kinetics (i.e., a Kd > IO-‘M), then use the procedure of Section 3.2.; otherwise, use the procedure of Section 3.3 3. See Note 10 for data analysis.

3.5. ~%]GTPyS/~H]Antagonist Simultaneous Measurement and Functional Effect (6)

Dual Label Binding Assay: of Agonist Binding

Figure 2 shows an example. 1. Prepare tubes with 10 pL agonist and 1-mL membranes containing GDP. 2. Add 10 pL of [3H]antagonist radioligand and incubate at 30°C until bmding is at equilibrium. 3. Add 10 pL diluted [35S]GTPyS to each tube and continue the incubation for 30 min. 4. Filter over wetted (with water) filters and wash twice with 3 mL ice-cold washing buffer (see Notes 5 and 13).

Agonist-Stimulated

111

r5S]GTPyS Binding Binding of rH’jNMS ml receptors

Binding of [35S]GTPyS to mlCH0 membranes

to

40000u 0 =:

30000

G 5i gz-

20000

:

:I

/./

-00

b------J -6 -7 -6

E 8

1-L

-5

-4

-3

01.1,

I

-ea

0 + 10 pg/rnl saponin

-7

,

-6

,

-5

I

-4

I-3

[ACh] log M

IAChl log M 0 Alone

-6

0 Alone

0 + 10 pg/ml saponin

Fig. 2. Dual-label experiment to measure simultaneously the effect of saponin on ACh stimulation of [35S]GTPyS binding to G proteins and inhibition of antagonist radioligand (3H-NMS) binding to muscarinic ml receptors in CHO cell membranes. 0.1 @4 GDP was present. The assay was conducted in duplicate. 5. Distribute the filter disks to scintillation vials and add scintillation fluid (see Note 11). 6. Determine the radioactivity in each filter using the dual 3H/35S label program provided with the scintillation counter (see Notes 11 and 12). 7. See Note 13 for problems of contamination.

3.6. p5S]GTPyS Binding Assay: lmprowing the SignaMo-Noise Ratio 1. Optimize the assay as described in Notes 2 and 5. Check incubation time and temperature. 2. Purify the plasma membrane preparation with sucrose density gradient centrifugation (e.g., ref. 7). 3. Include 10 pg/rnL saponin in the assay (-1: 1 saponin:protein). We have found (ref. 8 and S. Lazareno, unpublished observations; see Fig. 2) that this increases the signal-to-noise ratio without perturbing the pharmacological characteristics of the preparation, though possible effects of saponin on the pharmacological parameters being studied should be assessed.Other authors have used alamethicin as a permeabilizing agent (9). 4. Stop the reaction by adding high concentrations of unlabeled GTPyS (e.g., 10 @4) and competitive antagonist. Allow the label to dissociate for 45 min before filtration. This may yield a modest (lO--30%) increase in the signal-tonoise ratio.

112

Lazareno

4. Notes 1. [35S]GTPyS is currently supplied by NEN (New England Nuclear, Du Pont, UK) at a concentration of about 1V5M in a buffer containing 10 miV tricme and 10 mM dithiothreitol, pH 7.6. It should be diluted at least IO-fold in this buffer and stored at -70°C in aliquots sufficient for two or three assays, in order to minimize radiochemical decomposition. Accordmg to the volumes and dilutions recommended m this chapter, this will result in a concentration of 1 @4 dithiothreitol m the assay. 2. The precise concentrations of NaCl and MgCI,, and particularly of GDP, required for the optimal signal-to-noise ratio (maximal agonist-stimulated binding/basal binding) should be determined for each receptor-G protein preparation For some receptors, e.g., muscarimc ml receptors expressed m CHO cells, agonists stimulate [3sS]GTPyS binding in the absence of GDP, and mclusion of up to 0.1 @4 GDP in the [35S]GTP$S binding assay reduces basal binding without reducing stimulated binding. For other receptors, such as the muscarmic m2 receptor, GDP is absolutely required for agonist stimulation, and 1 @4 GDP was found to provide optimal results; 10 l.aV GDP was required with the adenosine At receptor (8,10). In all cases, sufficient bound counts must be obtained for accurate measurement, but not more than about 10% of added [35S]GTpYS should be bound, so less ml receptor membrane is used (5 pg protein/ml) than m2 receptor membrane (20 cc9/ mL). Note that GDP reduces agonist potency m the [35S]GTPyS binding assay (6). 3, The concentrated membranes may be stored at -70°C and later thawed for protein determination, dilution, and refreezing, without loss of stimulated [35S]GTPyS binding activity. 4, Because this is a binding assay, the variability between replicates is usually small, 2-5% of the mean counts, so duplicate measures are often sufficient. On the other hand, the stimulation of binding by agonist above basal values may be less than twofold, so triplicate or even quadrnphcate determinations may be necessary. 5. We find cold water to be acceptable for washing the filters. The filter blank, measured m the absence of membranes, is usually less than 0.5% of added label. Occasionally, however, much larger filter blanks occur, for reasons which are as yet unresolved. Large filter blanks may be reduced by washing in phosphate buffer. Nonspecific binding, measured in the presence of 10 @4 unlabeled GTPyS, is usually close to filter blank levels. 6. As far as possible, experiments should be designed so that all the data contributmg to an experimental question are obtained from a single filter. If this is not possible, then two or more filters should be used, each containing a single replicate of each experimental condition. In this way the variability between filters caused by small differences in washing procedure or incubation time is evenly dtstributed across all experimental conditions. In order to minimize possible position effects of the cell harvester, the second replicates may be filtered in reverse order. This is easily achieved by reversing the order in which samples are filtered, and then reversing the filter before distributing the filter disks to scintillation vials. 7. Filters dried before addition of scmtillation fluid can be counted immediately Counting wet filters will provide a good indication of the result of the assay, but

Agonist-Stimulated

p5S]GTPyS Binding

113

for accurate data the prepared vials should be left overnight and then inverted and shaken repeatedly to ensure that the water in the filter has completely dissolved in the scintillation fluid. 8. The affinity of a competttive antagonist is estimated in functional studies by constructing agonist concentration-effect curves, alone and in the presence of varrous fixed concentrations of antagonist, and analyzing the data with Schild analysis (11). This design allows the detection of any changes in basal activity, the E,,,,,, or the shape of the agonist curve in the presence of antagonist. If it can be assumed that the antagonist does not alter Em,, or agonist slope, it is simpler and more efficient to estimate antagonist affinity with this assay using an inhibition curve design. In this case, a minimum of two concentration-effect curves is required: an agontst curve alone, and an antagonist curve in the presence of a fixed agonist concentratton. The effect of the antagonist on basal activity should also be measured, though this mformation may not contribute to the data analysis. 9. Although the data may be analyzed using linear transformations and even “by eye ” mterpolations from graphs (12,13), ideally the data should be analyzed using nonlmear regression analysis. The manual provided with the Prism program (GraphPad) contams an introduction to nonlinear regression and other useful topics the relevant chapters are freely available on the World Wide Web (http:// www.graphpad.com). Some programs (e.g., Prism, see ref. 14) can only handle one independent vanable, e.g., drug concentration, Others (e.g., SigmaPlot [Jandel] and modern spreadsheets [15/) can handle two or more independent variables, e.g., agonist and antagonist concentrations. Equatrons containmg one independent variable are marked (*); those containing two independent variables are marked (**). Schild plot designs can be analyzed m the traditional way (I I) or by flttmg the complete data set, together with agonist [A] and antagonist [B] concentrations, directly to the integrated logisticSchild equation (22,13): Effect =

Ema, - basal

+ basal

(1) t**>

to yield estimates of basal activtty, Emax, agonist ECSo, agonist slope factor b, antagonist dissociation constant &, and Schild slope s. Inhibition curve designs can be analyzed in three ways (12,13). a. Using a SigmaPlot-type program, tit the complete data set to Eq. (1). b. Using a Prism-type program: i. Fit the agonist curve to a logistic function; Eq. (2). Effect = C-m - basal + basal

1+ IWd lb PI

114

Lazareno 11. Fit the antagomst curve to Eq. (I) (*), with [A] set to the fixed agonist concentration, and basal, Emax, EC,,, and b set to the values obtamed from the analysis of the agonist curve c. Using a Prism-type program. i Fit both curves to logistic functions; Eq. (2) ii. Calculate Kb, the antagonist dissociation constant, by inserting the values of the fixed agonist concentration ([A]), the EC,, and slope (b) of the agonist fit and the IC,, from the antagomst fit, mto the functional ChengPrusoff equation (14).

This analysis assumes that the antagonist Schild slope is 1. 10 If the agent does not affect basal activity, Emax, or the shape of the agonist curve,

then the complete data set, together with the concentrations of agonist [A] and allosteric agent [Xl, are fitted to the equation: E max- basal

Effect =

1 + IW5d [A]

.

1 + [x]/K,y

+ basal b

(4) (**)

1 +a$Z-/Z&j

to yield estimates of the dissociation constant of the allosteric agent, Kx, and the cooperativity with the agonist, c1 (5). 11. Filters that are dried before addition of scintillation fluid can be counted immediately. Otherwise, it is important that all the water in the filter disk be dissolved m the scintillation fluid (see Note 7) 3H is much more quenched by water then 35S; if the filter disk is wet, the dual label program will not function correctly, and the results will be qualitatively mcorrect. 12. Ideally, one isotope should not give more than about 30 times the counts given by the other isotope 13. Two types of contamination

may occur if a cell harvester is used with both r3H]antagonist and [35S]GTPyS. a. The tubing will be contaminated with 35S, which will leach out and contaminate 3H assays. We have observed contamination of over 1000 dpm per sample. The contamination is progressively reduced during a day as more 3H assays are filtered, but is higher at the start of the next day. We cope with the problem by routinely countmg 3H with a dual-label program. b. Serious filter blank problems occur with [35S]GTPyS if the filter is exposed to even very small amounts (1 part in lo7 w/v) of polyethyleneimme (PEI). In many radioligand binding assays, filters are soaked with 0.1% PEI to reduce the binding of positively charged radioligands The filtration apparatus will become contaminated with PEI. and r3’S1GTPvS binding assavs usins the

Agonist-Stimulated /%]GTPyS Binding

115

same apparatus will often have some spuriously high readings, especially with the first filter of the day. The solution is to filter washing buffer or water through two filters (or much cheaper, paper towels) before filtering [35S]GTPyS.

References 1. Neer, E. J. (1995) Heterotrimeric G proteins organizers of transmembrane srgnals. Cell 80,249-257. 2 Hilf, G., Gierschik, P., and Jakobs, K. H. (1989) Muscarinic acetylcholine receptor-stimulated binding of guanosine 5’-O-(3-thiotriphosphate) to guanine-nucleotide-binding proteins m cardiac membranes. Eur. J. Bzochem 186, 725-731. 3. Wieland, T. and Jakobs, K. H. (1994) Measurement of receptor-stimulated guanosine 5’-0-(y-thro)tnphosphate binding by G proteins. Methods Enzymol. 237,3-13. 4. Sweeney, M. (1995) Measurement of the GTPase activity of signal-transducing G-proteins in neuronal membranes, in Methods in Molecular Bzologv, vol. 41 (Kendall, D. A. and Hill, S. J , eds.), Humana, Totowa, NJ, pp. 5 1-61. 5. Lazareno, S. and Brrdsall, N. J. M. (1995) Detection, quantitatron, and verrtication of allosteric interactions of agents with labeled and unlabeled hgands at G protein-coupled receptors: interactions of strychnine and acetylcholine at muscarinic receptors. Mol Pharmacol. 48,362-378. 6. Lazareno, S., Farrres, T., and Birdsall, N. J. M. (1993) Pharmacologrcal characterization of guanine nucleotide exchange reactrons m membranes from CHO cells stably transfected with human muscarinic receptors ml-m4. Lrfe. Scz. 52, 449456 7. Hilf, G. and Jakobs, K. H. (1989) Activation of cardiac G-proteins by muscarinic acetylcholine receptors: regulation by Mg2+ and Na+ ions. Eur J. Pharmacol 172,155-163

8. Cohen, F. R., Lazareno, S., and Birdsall, N. J. M. (1996) The effects of saponin on the binding and functional properties of the human A, adenosine receptor. Br. J. Pharmacol. 117,152 l-l 529. 9. Hilf, G. and Jakobs, K. H. (1992) Agonist-independent inhibition of G protein activation by muscarmic acetylcholme receptor antagonists m cardiac membranes Eur. J. Pharmacol

225,245-252.

10. Lorenzen, A., Fuss, M., Vogt, H., and Schwabe, U. (1993) Measurement of guanine nucleotide-binding protein activation by A, adenosine receptor agonists in bovine brain membranes: stimulation of guanosine-5’-0-(3-[35S]thlo)triphosphate binding. Mol. Pharmacol 44, 115-123. 11. Kenakin, T. (1993) Pharmacologic analysis of drug-receptor interaction. Raven, New York. 12. Lazareno, S. and Birdsall, N. J. M. (1993) Estimation of competitive antagonist affinity from functional inhibition curves using the Gaddum, Schrld and ChengPrusoff equations. Br J. Pharmacol, 109,1110-l 119.

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13, Lazareno, S. and Birdsall, N. J. M. ( 1993) Estimation of antagonist Kb from inhibition curves in functional experiments: alternatives to the Cheng-Prusoff equation. Trends Pharmacol. Sci. 14,237-239. 14. Lazareno, S. (1994) Graphpad prism (Version 1.02): Software review. Trends Pharmacol. Sci. 15,353,354.

15. Bowen, W. P. and Jerman, J. C. (1995) Nonlinear regression using spreadsheets. Trends Pharmacol

Sci. 16,413-417.

16. Leff, P. and Dougall, I. P. (1993) Further concerns over Cheng-Prusoff analysis. Trends Pharmacol Ser. 14,110-l 12.

Autoradiographic Visualization in Brain of Receptor-G Protein Coupling Using [35S]GTPyS Binding Laura J. Sim, Dana E. Selley, and Steven I?. Childers 1. Introduction 1.1. Autoradiographic Localization of Receptor-G Protein Coupling Localization of receptors m brain sections using autoradtographlc detection of radioligand binding has been an important technique in the neuroanatomical identification of a large number of neurotransmitter receptors. However, receptor autoradiography provides little information regarding the functional relevance of these sites and, in fact, does not establish which of the labeled receptor sites are actually coupled to intracellular signaling mechanisms. Fortunately, with the family of G protein-coupled receptors (GPCR), signal transduction is mediated at the level of the transducer itself (i.e., at the point at which receptors activate the a-subunits of G proteins to bind guanosine S-triphosphate [GTP]). The development of an assayfor agonist-stimulated [35S]guanylyl-5’-O-(y-thio)-triphosphate ([35S]GTPyS) binding, originally developed for receptors in isolated membranes, has provided an excellent opportunity to apply this process to brain sections, thus allowing the visualization of receptor-activated G proteins in specific brain regions. This chapter describes the recent development of in vitro autoradiography of receptor-stimulated [35S]GTPyS binding in brain sections. One of the keys to the success of this technique is the use of a large excess of guanosine S-diphosphate (GDP) to inactivate G protem a-subunits and reduce basal [35S]GTPyS binding. Using this technique, [35S]GTPyS binding, stimulated by multiple GPCRs, can be localized in adjacent sections using a single From: Methods II) Molecular Biology, vol 83 Receptor S/gnat Transduction Edlted by R A J Chalks Humana Press Inc , Totowa, NJ

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Protocols

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radtohgand, [35S]GTPyS. Moreover, since the technique utilizes [35S] as a radiolabel, film exposure is much shorter than traditional receptor autoradiography using [3H] radioligands. Most important, this technique provides a fundamentally different type of mformation than traditional receptor autoradiography, since it measures activation of an mtracellular signal transduction pathway and provides functional information regarding these systems m brain. Thus, the activity of receptor agonists and antagonists can be differentiated, and basic pharmacological questions can investigated with neuroanatomical precision. 1.2. History of Receptor Localization

and G Protein Coupling

The first studies on receptor localization were performed by injecting radiolabeled ligands in vivo (1,2). This method has several disadvantages, mcluding high nonspecific binding, restriction to examining only one ligand per brain, and the use of large amounts of radioligand. The development of in vitro receptor autoradiography (3,4), based on receptor-binding assaysm membranes (5), solved many of these problems. In addition to the ability to localize receptors in an anatomically specific manner, this technique allows the examination of multiple receptor systems in the same brain. Furthermore, the assay is not limited to radioligands that are stable for in vivo procedures, and allows in vitro optimization of assay conditions for specific receptors. However, receptor autoradiography is limited to the detection of ligand binding sites and provides no information regarding the functional activity of receptors. A large number (at least 80%) of neurotransmttters and hormones produce their biological effects via GPCRs (6) and the cycle of receptor-G protein activation has been extensively reviewed (6,7). Each G protein is a heterotrimer composed of three subunits, a-, /3-,and y-. The binding of agonist activates its receptor; this activation changes the conformation of the G protein, increasing the affinity of the a-subunit for GTP and decreasing its affinity for GDP. GTP binding activates the a-subunit, and decreases its affinity for Py, causing l3yto dissociate from a. The receptor and a then dissociate, enablmg a and l+yto interact with effecters (6-10). The a-subunit spontaneously inactivates by intrinsic GTPase that hydrolyzes GTP to GDP, increasing the affinity of o. for l3r. The a-subunit reassociates with Pr, completmg the cycle. The reaction is catalytic, since each receptor can activate multiple G proteins, resulting in amplification of the receptor signal into an intracellular response (II, 22). Receptor activation of G proteins in isolated membranes has traditionally been measured by receptor-stimulated low K, GTPase activity (13-15). However, this method can be misleading, since factors that affect GTP hydrolysis and not G protein activation will affect the results (16). The GTPase assay is also a rather indirect measure of G protein activity, since it measures hydroly-

Autoradiography

of GPCR Using /3]GTPyS

119 Excess

GDP

/ G PROTEINS

Agonist [=S]GTP-/S or GTP

R

1

GTP/

ACTIVATED

[%]GTPyS

G PROTEINS

EFFECTOR

Fig. 1. Schematic diagram of the mechanism of agonist-stimulated [35S]GTPyS binding. (A) Addition of excess GDP shifts the G proteins into the inactive state. (B) Agonist binds to the receptor, which increases the affmity of the G protein for GTP or [35S]GTPyS. (C) Activation of the G protein by GTP activates effecters or; (D) Activation of the G protein by [35S]GTPyS provides labeled G protein.

sis, which is the inactivation reaction of the G protein cycle. Receptor-stimulated [35S]GTPyS binding is a more direct assay of receptor activation of G proteins, since it measures the activation reaction of the cycle (i.e., the exchange of bound GDP for GTP [or [35S]GTPyS]). This technique has been used in biochemical studies of purified and reconstituted systems (11,17,18). More recently, the [35S]GTPyS technique has been applied to isolated cardiac (19), brain (20), and cultured cell (‘22,221 membranes to measure specific receptor-stimulated G protein activity. The stimulation of [35S]GTPySbinding to G proteins by receptor agonists is based upon the G protein activation cycle discussed above (see Fig. 1). Initially, an excess of GDP is added to the assay to shift the G protein into the

720

Sim, Selley, and Childers

inactive state. [35S]GTPyS and agonist are then added to activate the G protein coupled to the receptor of interest. Receptor activation decreases the a-subunit’s affinity for GDP and increases its affinity for GTP, so that the G protein binds GTP, or [35S]GTPyS. In vivo, the a-subunit GTPase hydrolyzes GTP to GDP; however, [35S]GTPyS is resistant to hydrolysis and remains bound. The increase in bound [35S] induced by agonist can then be measured by autoradiography or liquid scintillation spectrometry.

2. Materials 1. Assay buffer: 50 mMTris-HCl, containing 3 mM MgCI,, 0.2 mM EGTA, 100 mM NaCl, pH 7.4. Sodium (Na) is required in the [35S]GTPyS assay to decrease basal binding (19,23). Sodium has also been shown to decrease basal G protein activity m the GTPase assay (16,24-26). The reason for this effect is, at least in part, that sodium decreases spontaneous G protein activation produced by unoccupied receptors (23,26). The omission of sodium from the [3sS]GTPyS autoradlographlc assay results in a high level of basal [3SS]GTPyS binding above which agonistsimulated [35S]GTpVS binding is undetectable. Magnesium has a biphasic effect on G protein activity (7,27). Low (high nanomolar to low micromolar) concentrations of magnesium are required for hydrolysis of G,-GTP and high (high micromolar to low millimolar) concentrations promote agonist-stimulated G protein activation, so the magnesmm concentration must be optimized to achieve maximal [3sS]GTPyS binding. 2. Supplements to assay buffer: The assay buffer may be modified to optimize agonist-stimulated [35S]GTPyS binding for some agonists. For instance, 0.5% bovine serum albumin is added to the incubation media for the cannabinoid agonist WIN 55212-2. Protease inhibitors (10 &/mL of a solution containing 0.2 mg/mL each of bestatin, leupeptin, pepstatin A, and aprotinin) may also be added to the preincubation buffer for endogenous peptides. The pH of the buffers is especially important in the [3SS]GTPyS autoradiographic assay, since tissue may be damaged by acidic or basic pH 3. Wash buffer: 50 mMTri.s-HCl, pH 7.0 at 25°C 4. Nucleotides: A large excess of GDP must be added to the [3sS]GTPyS assay to decrease basal binding of [35S]GTPyS (see Note 1). Although other nucleotides may also reduce basal binding, GDP is the most effective in decreasing basal binding tihile allowing receptor stimulation of [35S]GTPyS bindmg (Z9). GDP decreases basal [35S]GTpYS binding by shifting the G protein into the inactive (GDP-bound) state. In isolated membranes, 10-100 @4GDP is sufficient to allow for significant agonist-stimulated [35S]GTPyS binding. GDP is especially important in the [35S]GTPyS autoradiographic assay in brain sections (28). In this case, 1-2-M concentrations of GDP are necessary to detect agonist-stimulated [35S]GTPyS binding over basal [35S]GTPyS binding (see Fig. 2). The reason that such a high concentration of GDP is required in the brain sections is not completely understood, but may be because of the ability of GDP to effectively pen-

Autoradiography

of GPCR Using r%]GTPyS

10 ylM GDP

100 pM GDP

1000 PM GDP

121 2000ylM

GDP

- DAMGO

- DAMGO

- DAMGO

- DAMGO

+ DAMGO

+ DAMGO

+ DAMGO

+ DAMGO

Fig. 2. Effect of GDP on basal and mu opioid-stimulated [35S]GTPyS binding in rat brain sections at the level of the thalamus. Brain sections were incubated with varying concentrations of GDP in the presence or absence of DAMGO. Mu opioid-stimulated [3sS]GTPyS binding is not detected until the GDP concentration reaches l-2 mM. en-ate intact sections, or be caused by the level of total protein in sections. There are several ways to minimize the use of GDP to decrease the cost of the assay (see Note 1). The storage of both [3sS]GTPyS (New England Nuclear, Boston, MA) and unlabeled GTPyS is an important consideration, since GTPyS is relatively unstable. [‘%]GTPyS is diluted 1:lO in H,O and stored at -40°C. Appropriate dilutions for assays are made from this stock on the day of the assay; these diluted assay solutions are never stored for future use. Unlabeled GTPyS is stored desiccated at -20°C. Stock solutions are made and stored in aliquots at -80°C until use. 5. Receptor agonists: The choice of agonists for the [35S]GTPyS assay is limited mainly by the specificity and metabolic stability of the agonist, as well as its ability to act as a full agonist (see Notes 2 and 3). 6. Autoradiographic film: Several types of autoradiographic film may be used, since [35S] is a relatively high-energy radioisotope. Our laboratory uses Reflections@’ (New England Nuclear) film because of its low background, but other types of film appropriate for [35S] have also been used successfully.

3. Methods 3.1. Autoradiography 1. Animals are sacrificed by rapid decapitation. Brains are removed and slowly immersed in isopentane maintained at -35’C on dry ice. After 3-5 min, the brains are removed from the isopentane with cold forceps and placed on dry ice for

122

2

3.

4.

5.

6

7.

8.

Sim, Selley, and Childers 5 mm to evaporate remaining isopentane. The brains are wrapped m cold foil, placed in sealed polypropylene containers, and stored at -80°C until cuttmg Brains are cut mto 209.m sections on a cryostat maintained at -20°C and sections are thaw-mounted onto gelatin-subbed slides. Slides are collected in racks in a humidified chamber on ice, desiccated under vacuum, and stored overnight at 4°C. The next day, shdes are placed in shde boxes with desiccant pellets, and stored at -80°C until use. On the day of the assay, slides are removed from the freezer and brought to room temperature under a cool air dryer for about 30-60 min. Slides are transferred into slide racks and equihbrated m assay buffer (see Section 2.) for 10 mm at 25’C. Incubations are performed in a water bath maintained at 25”C, so that the temperature remains constant Shdes are transferred to mailers (5 slides) or polypropylene coplin jars (10 slides) and incubated for 15 min in assay buffer containing l-2 &GDP (see Note 1). Slides are then transferred to mcubatlon media that contains 0.04 nA4[35S]GTPyS, l-2 mA4GDP and agonist in assay buffer, and incubated for 2 h at 25°C Basal binding is measured in the absence of agonist (1.e , with GDP and [35S]GTPyS) and nonspecific binding is assessed in the presence of 10 uh4 GTPyS For final rinses, slides are transferred into slide racks and rmsed in stainmg dishes Slides are rinsed twice for 2 min each in Tris buffer (see Section 2.) on ice and once for 30 s in deionized H,O on ice For diagnostic purposes, sections can be wiped from the slide with filter paper and counted in a scintillation counter. For autoradiography, slides are dried well under a cool stream of air (30-60 mm), then covered and dried overnight at room temperature. When using Reflections film, slides are generally exposed for 48 h, although longer exposures (4-7 d) may be necessary for less efficacious agonists. Exposure time may also be modified if a different type of film IS used, dependmg on the sensitivity.

3.2. Analysis

of Data

1. In our laboratory, films are digitized with a Sony XC-77 video camera and analyzed usmg the NIH IMAGE program for Macintosh computers. The regions measured are selected based on interests of the particular study and determmation of regions with relatively high levels of agonist-stimulated [35S]GTPyS binding. 2. It is important to select regions with optical densities that fall within the limits of the standard (i.e., that are not too high or too low to be measured reliably). The film exposure time may be modified so that the sections are not over- or underexposed Images are captured by averaging 16 mdividual frames of the image to provide a more accurate density measurement. 3. Quantification is obtained by including [14C] microscales in the cassette. Since densitometric analysis is performed using [14C] standards, a bram paste standard assay has been performed to determine the correction factor necessary to calculate nCi/g for [35S] from nCi/g for [ 14C].

Autoradiography

of GPCR Using P5S]G TPyS

123

4. Brtefly, known amounts of [35S]GTPyS are added to homogenized bram (bram paste), which is frozen into a block and sectioned at 20 pm. Sections are collected to measure wetght, radioactivity by scmtillation counting, and densitometry by exposure to film. 5. From these values, a correction factor is obtained to transform the nCi/g based on [ 14C!]standard densitometry mto nCi/g for [35S]. In our laboratory, the correction factor for [14C] to [35S] in 20-pm brain sections is. [35S] = 0.697 x ([14C]) - 96.4. It is important to note, however, that this correction formula will vary based on the tissue and type of film used. 6. For data analysis, sections are assayed in duplicate or triplicate m brains from at least five animals. Results are expressed as both net-sttmulated [35S]GTPyS binding in nCi/g (agonist-stimulated [35S]GTPyS binding minus basal binding) or percent stimulation (net binding divided by basal binding x 100).

3.3. General Applications

of the Method

1. Advantages: [35S]GTPyS autoradiography provides neuroanatomlcal and pharmacological data regarding receptor-G protein function. This technique differentiates between agonist and antagonist effects of drugs at specific receptor sites, which greatly increases the power of the technique. Moreover, this technique can be used to address differences m efficiency of coupling of receptors to G protems in specific brain nuclei. Although the basic methodology of [35S]GTPyS autoradiography is similar to that of traditional receptor autoradtography, there are several important differences between these techniques. Perhaps most important 1s the concept that [35S]GTPyS autoradrography demonstrates functional activity at the level of signal transduction, rather than binding at the level of the receptor [35S]GTPyS autoradtography also has several technical advantages, mcluding short film exposure, the ability to examine multiple receptor systems m adjacent sections, and the ability to use any available unlabeled agonist to activate the receptor, The use of unlabeled ligand is especially important for newly Isolated or synthesrzed hgands, for which radiolabeled ligands are not commercially available. For example, [35S]GTPyS autoradiography has been used to map the distribution of opioid receptor-like (ORL-1) receptor-activated G proteins (34), (see Fig. 3). ORL-1 peptide was synthesized based upon the published sequence (35,36), and used in the [35S]GTPyS assay to obtain a neuroanatomical localization of this receptor, although a radiolabeled peptide was not yet commercially available. 2. Limitations: Since the [35S]GTP$S assay examines function at the level of receptor-G protein coupling, many of the limitations of the technique result from functional properties of the system. Perhaps the most obvious limitatron of [35S]GTPyS autoradiography is the difficulty in detecting certain classes of receptors because of low receptor density and/or low efficiency of couplmg, as discussed above. Thus far, the assay has not been applicable to G,-coupled sys-

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Basal

DAMGO Fig. 3. Rat brain sections at the level of the caudate-putamen comparing ORL-1 peptide- and mu opioid-stimulated [35S]GTPyS binding. Mu opioid-stimulated [35S]GTPyS binding is seen in the patches of the caudate-putamen. In contrast, ORL1 peptide-stimulated [35S]GTPyS binding is absent in the caudate-putamen, but high in the cortex.

terns in brain membranes or sections. This is because the relative abundance of G proteins in the brain is G, > Gi > G, (37), so that the level of G, may be too low to produce detectable [35S]GTPyS binding. However, [35S]GTPyS binding stimulated by G,-coupled receptors can be measured in other systems. Prostaglandin El-stimulated [35S]GTPyS binding is measurable in NGlOS-15 cell membranes and isoproterenol-stimulated [35S]GTPyS binding is detectable in turkey erythrocyte membranes (38). Another limitation is the specificity of the technique for individual G proteins. Although the [35S]GTP-yS binding assay is highly specific for receptor type (by the use of selective agonists), there is currently no method to determine which type of G protein(s) the receptor activates in the tissue section. Thus, the [3SS]GTPyS binding signal results from the activation of the pool of G proteins coupled to the receptor of interest, which may be a heterogeneous G protein population.

3.4. Effects of Chronic Agonist

Treatment

An important application of the [35S]GTpVS assay, and particularly [35S]GTPyS autoradiography, is the investigation of receptor-G protein function after chronic drug treatment. The first study in which [35S]GTPyS binding was used to examine the effects of chronic drug treatment was performed using chronic morphine administration in rats (39). Using [35S]GTPyS autoradiography, chronic morphine treatment was shown to have no effect in telencephalic and diencephalic mu receptor-containing nuclei, including the striatum and thala-

Autoradiography

125

of GPCR Using P%]GTPyS BASAL

DAMGO

CONTROL

CHRONIC MORPHINE Fig. 4. Autoradiograms of brain sections from control and chronic morphine-treated rats. A high level of mu opioid-stimulated [35S]GTPyS binding is found in the parabrachial nucleus in the lateral portion of the section. This mu opioid-stimulated G protein activity is significantly decreased following chronic morphine administration.

mus. However, specific decreases in mu opioid-stimulated [35S]GTPyS binding were observed after chronic morphine treatment in specific brainstem nuclei, including the dorsal raphe nucleus, locus coeruleus, parabrachial nucleus, and commissural nucleus tractus solitarius (Fig. 4). These areas have been implicated in autonomic functions of opioids, which are predominant during the development of dependence and withdrawal. In contrast, acute morphine treatment had no significant effect on [35S]GTPySbinding in any of these brainstem nuclei, indicating that these effects were specifically produced by chronic agonist treatment. More recent studies have examined changes in cannabinoid-stimulated [35S]GTPyS binding after chronic A9 tetrahydrocannabinol (A9-THC) treatment of rats (40). In this study, chronic A9-THC treatment produced significant decreases in cannabinoid-stimulated [35S]GTPySbinding. However, unlike the results of the chronic morphine study, in which effects on G protein activation were confined to specific brainstem nuclei, changes in cannabinoid-stimulated [35S]GTPyS binding were widespread, throughout regions including the hippocampus, entorhinal cortex, perirhinal cortex, caudate-putamen, globus pallidus, cerebellum, and substantia nigra. The magnitude of these changes

Sim, Selley, and Childers

126 BASAL

WIN 55,212-2

CHRONIC Ag-THC

Fig. 5. Autoradiograms of brain sections from control and chronic A9-THC-treated rats. Chronic A9-THC treatment results in decreases in cannabinoid-stimulated [35S]GTPyS binding throughout the brain. In this section, decreased cannabinoid-stimulated [35S]GTPyS binding is seen in the cortex, caudate-putamen, and hippocampus.

was dramatic; decreases in cannabinoid-stimulated [35S]GTPyS binding were at least 50% in most regions (Fig. 5). These studies, together with the chronic morphine experiments cited above, demonstrate that [35S]GTPyS autoradiography can be used to detect changes in G protein activity after chronic drug treatment. Thus, in addition to localization of receptor-activated G proteins, [35S]GTPyS autoradiography can be used to detect changes following experimental treatments or in models of disease states. This aspect of the technique makes [35S]GTPyS autoradiography applicable to a large number of different types of studies. 4. Notes 1. Assay GDP concentrations: A high concentration (l-2 m&Q of GDP is used in the autoradiographic assay in the 15min preincubation, as well as the final incubation with agonist and [35S]GTPyS. The final concentration of GDP to use in the assay must be determined empirically. GDP concentrations below 1 mM are not recommended, since basal levels of [3SS]GTPyS binding become too high to detect agonist-stimulated r3%]GTPyS binding. High (2 mM) concentrations of GDP decrease both basal and stimulated labeling, to produce a lighter image, and maximize the percent stimulation by agonist. Lower (1 mM) concentrations of GDP produce higher labeling and faster exposure times, but lower the percent

Autoradiography of GPCR Using r”S]GTPyS

127

stimulation by agomst. The GDP premcubation solution may be discarded and a new incubation solution containing GDP, [35S]GTPyS, and agonist used for the assay However, to reduce the cost of GDP, [35S]GTPyS, and agomst can be added to the premcubation solution. In this case, the solution must be mixed well, which is done by removing the slides and carefully mixing the solution It is also possible to perform subsequent runs of the assay, so that the same incubation mixture is used for multiple sets of slides. In our laboratory, the incubation solution is used a maximum of two times. The relationship between protein concentration and GDP concentration is an important consideration in the [35S]GTPyS binding assay. In assays of receptorstimulated [35S]GTPyS bmding to isolated membranes, increasing protein concentrations requires concomitant increases in GDP concentrations because of the increased level of basal [35S]GTPyS binding. In brain membrane assays using 10-20 @I GDP, 2-l 5 pg protein per assay tube are generally used, depending on the region examined. In contrast, in autoradiographic assays, each 20-pm rat brain section (at the level of the caudate-putamen) contains approx 175 pg protein This may explain why relatively high GDP concentrations are required m sections relative to membrane assays to reduce basal [35S]GTPyS binding. 2. Methods development: When establishing a new protocol, each prehmmary experiment should contain a positive control, i.e., a condition that duplicates a published result. Both mu opioid (e.g., using 3-10 @4 DAMGO) and GABA, (e.g., using 300 pd4 baclofen) agonists provide excellent signals and characteristic labeling patterns. The [3sS]GTPyS autoradiographic assay is also best performed in conlunction with [35S]GTPyS membrane binding assays (28) to determine optimal conditions for the assay. This IS especially important m determining the concentration of agonist to use for maximal stimulation of [35S]GTPyS binding. 3. Pharmacological validation: Since [35S]GTPyS autoradiography depends on agonist-receptor interactions, several pharmacological criteria must be met to verify the specificity of the assay. First, agonist-stimulated [35S]GTPyS binding must be blocked by the appropriate antagonist (Fig. 6) For example, in the mu opioid receptor system, stimulation of [35S]GTPyS binding was blocked by the opioid antagonist naloxone, and WIN 55212-2-stimulated [35S]GTPyS binding via cannabinoid receptors was blocked by the cannabinoid antagonist SR 141716A (28). Agonist-stimulated [35S]GTPyS binding must also be concentration-dependent and saturable with regard to agonist. This has been demonstrated in both isolated membranes and tissue sections (28). In isolated membranes, some agonists produce an artifactual overshoot of [35S]GTPyS binding when used at high concentrations (i.e., over 30 CIM). Thus, in determining the agonist concentration to use for maximal stimulation of [35S]GTPyS binding, it is important to verify that the response is antagonist-reversible (i.e., receptor mediated). Finally, since agonist-stimulated [35S]GTPyS binding results from receptor activation of G proteins, the anatomical distribution of agonist-stimulated [35S]GTPyS bmding should correlate with that of radiohgand bmding. This has been true for the

Sitn, S&y,

DAMGO

WIN

55212-2

DAMGO

WIN

and Childers

+ NALOXONE

55212.2

+ SR 141716A

Fig. 6. Autoradiograms demonstrating antagonist reversal of mu opioid- (top) and cannabinoid- (bottom) stimulated [35S]GTPyS binding. Mu opioid- (using 3 @4 DAMGO as an agonist) stimulated [35S]GTPyS binding is found in the caudate-putamen, thalamus, and periaqueductal gray and is eliminated by the mu antagonist naloxone (0.3 llM). In a more ventral section, cannabinoid-stimulated [35S]GTPyS binding (using 1 pA4 WIN 552 12-2 as an agonist) is seen in the substantia nigra and is blocked by the addition of the cannabinoid antagonist SR 141716A (0.3 p&Q

receptor systems examined thus far, including cannabinoid, GABA,, ORL- 1, and p, 6, and K opioid (29-33). However, since the receptor-G protein interaction is catalytic, the relative levels of agonist-stimulated [3sS]GTPyS binding may not correlate with receptor density. 4. Troubleshooting: The majority of technical problems encountered thus far have resulted from improper storage of tissue or nucleotides. Tissue sections on slides for [35S]GTPyS autoradiography should be stored at -80% until use. Storage at higher temperatures, or freeze-thawing the sections, decreases the [35S]GTPyS signal and may eliminate agonist stimulation of [35S]GTPyS binding. The temperature is also a factor during the collection of sections from the cryostat. It is best to collect slides on ice, because decreased [3sS]GTPyS binding may result if slides are kept at room temperature for a prolonged time. As discussed in Materials, GTPyS is a rather unstable compound, so that improper storage may result in degradation of the compound. Assay parameters that may be adjusted to maximize the agonist-stimulated [35S]GTPyS signal are the concentration of [35S]GTPyS, incubation time and temperature, and film exposure time. One of the striking findings in comparing the results of [3SS]GTPyS autoradiography with that of traditional receptor autoradiography was the fact that qualitatively, the neuroanatomical distribution of agonist-stimulated

Autoradiography

729

of GPCR Using /W]GTPyS

WIN 55,212-P-STIMULATED [35S]GTPyS BINDING

[3H]WIN

55,212-2

BINDING

Fig. 7. Autoradiograms comparing cannabinoid-stimulated [35S]GTpYS binding and cannabinoid receptor binding in horizontal sections of the rat brain. Although cannabinoid binding sites and cannabinoid-activated G proteins exhibit identical neuroanatomical distributions, the relative levels of activity do not correlate in all regions. [35S]GTPyS binding closely paralleled that of receptor binding sites. However, from a quantitative perspective, interesting differences have emerged. One example is the cannabinoid receptor system, in which the level of agoniststimulated [35S]GTPyS binding did not completely correlate with reported receptor densities (Fig. 7). In this case, although there were lo-fold more cannabinoid than opioid binding sites in the striatum, the level of opioid-stimulated [35S]GTPyS binding was comparable to that of cannabinoid-stimulated [35S]GTPyS binding (28). Catalytic amplification factors were calculated for each receptor, based on receptor binding and G protein Scatchard analysis in isolated membranes, and showed that each cannabinoid receptor activated three G proteins, whereas each p and 6 opioid receptor activated 17-20 G proteins. Therefore, opioid receptors in the striatum are more catalytically efficient than cannabinoid receptors. This finding may have important technical implications regarding the [35S]GTPyS binding assay. Receptor density and catalytic activity of the system are factors that determine the magnitude of the [35S]GTPyS signal. Thus, it may be difficult or impossible to detect agoniststimulated [35S]GTPyS binding in a system with low receptor density and/or low catalytic activity.

References 1. Kuhar, M. J. and Yamamura, H. I. (1975) Light autoradiographic localisation of cholinergic muscarinic receptors in rat brain by specific binding of a potent antagonist. Nature 253,560,56 1. 2. Pert, C. B., Kuhar, M. J., and Snyder, S. H. (1975) Autoradiographic localization of the opiate receptor in rat brain. Life Sci. 16, 1849-l 854.

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3. Young, W. S and Kuhar, M J (1979) A new method for receptor autoradtography: [3H]opiotd receptors in rat brain Brain Res. 179,255-270. 4. Herkenham, M. and Pert, C. B (1980) In vitro autoradrography of opiate receptors m rat brain suggests loci of “optatergic” pathways. Proc. Nat1 Acad Scl USA 71,5532-5536. 5 Pert, C. B. and Snyder, S. H. (1973) Opiate receptor: Demonstration m nervous ttssue. Science 179, 101 l-1014. 6 Bu-nbaumer, L., Abramowitz, J., and Brown, A. M. (1990) Receptor-effector coupling by G proteins. Blochim. Bzophys Acta 1031, 163-224. 7. Gtlman, A. G (1987) G protems: transducers of receptor-generated signals. Annu Rev. Blochem. 56,615-649. 8. Htldebrandt, J D., Sekura, R D., Codma, J , Iyengar, R., Manclark, C. R , and Bnnbaumer, L. (1983) Strmulatron and inhtbitton of adenylyl cyclases mediated by distmct regulatory proteins Nature 302,706-709. 9. Brown, A M. and Brrnbaumer, L. (1990) Ionic channels and then regulatton by G protein subunits. Annu Rev Physlol 52, 197-2 13 10. Childers, S. R (1991) Oproid receptor-coupled second messengers. Lfe Scz 48, 1991-2003. Il. Asano, T , Pedersen, S. E., Scott, C. W., and Ross, E. M. (1984) Reconstitutton of catecholamine-stimulated binding of guanosme 5’-O-(3-throtriphosphate) to the stimulatory GTP-binding protein of adenylate cyclase Bzochemzstry 23, 5460-5467. 12 Gierschtk, P., Moghtader, R., Straub, C., Dieterich, K , and Jakobs, K. H (1991) Srgnal amphficatron m HL-60 granulocytes. evidence that the chemotactrc peptide receptor catalytically activates guanme-nucleottde-binding regulatory protems In native plasma membranes. Eur J. Bzochem 197, 725-732. 13 Cassel, D. and Selinger, Z. (1976) Catechoiamme-stimulated GTPase activity m turkey erythrocyte membranes. Blochzm Bzophys Acta 452,538-55 1 14. Koski, G and Klee, W. A (1981) Opiates inhibit adenylate cyclase by sttmulation of GTP hydrolysis. Proc. Nat1 Acad. SCL USA 78,4185-4189. 15 Selley, D. E. and Bidlack, J M. (1992) Effects of P-endorphin on Mu and Delta optoid receptor-coupled G protein activtty: Low-Km GTPase studies. J Pharmacol. Exp Ther 263,99-104. 16. Selley, D. E , Breivogel, C. S., and Childers, S. R. (1993) Modtfication of oprotd receptor-G-protein function by low pH pretreatment of membranes from NG10815 cells: Increase m opiotd agonist efficacy by decreased inacttvation of G-proteins. Mel Pharmacol 44,73 l-74 1, 17. Kurose, H., Katada, T , Haga, T , Haga, K., Ichiyama, A., and Ut, M. (1986) Functional interaction of purified muscarmic receptors with purified mhibitory guanine nucleotide regulatory proteins reconstituted in phosphohpid vesrcles. J. Blol Chem. 261,6423-6428. 18 Florro, V A. and Stemwers, P. C. (1989) Mechanisms of muscarimc receptor action on G, in reconstttuted phosphohpid vesicles. J Biol Chem. 264,3909-39 15.

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19. Hilf, G., Gierschik, P., and Jakobs, K. H. (1989) Muscarmic acetylcholme receptor-stimulated binding of guanosine 5’-0-(3-thiotriphosphate) to guanine-nucleotide-binding proteins m cardiac membranes. Eur J. Biochem 186,725-73 1. 20. Lorenzen, A., Fuss, M., Vogt, H., and Schwabe, U. (1993) Measurement of guarune nucleotide-bindmg protein activation by A, adenosme receptor agonists in bovine brain membranes stimulation of guanosine-5’-O-(3-[35S]thio)triphosphate bmdmg. Mel Pharmacol 44, 115-123 21 Lazareno, S., Fames, T., and Birdsall, N. J M. (1993) Pharmacological charactertzation of guanme nucleotide exchange reactions in membranes from CHO cells stably transfected with human muscarinic receptors MI-M4 tife sci 52,449+56. 22 Traynor, J. R. and Nahorski, S. R. (1995) Modulation by p-opioid agonists of guanosine-5’-O-(3-[35S]thio)triphosphate bmdmg to membranes from human neuroblastoma SH-SYSY cells. A4ol Pharmacol 47,848--854 23. Tian, W.-N., Duzic, E., Lamer, S. M., and Deth, R. C (1994) Determinants of az-adrenergic receptor activation of G proteins: evidence for a precoupled receptor/G protein state Mel Pharmacol. 45, 524-53 1. 24. Koski, G., Streaty, R. A , and Klee, W. A. (1982) Modulatton of sodium-sensitive GTPase by partial opiate agonists. J. Bzol. Chem. 257, 14,035-14,040. 25. Gierschik, P , Sidiropoulos, D., Steisslmger, M., and Jakobs, K. H. (1989) Na+ regulation of formyl peptide receptor-mediated signal transduction m HL60 cells. Evidence that the cation prevents activation of the G-protein by unoccupted receptors. Eur. J Pharmacol. 172,481-492 26. Costa, T., Lang, J , Gless, C., and Herz, A. (1990) Spontaneous association between opioid receptors and GTP-binding proteins in native membranes: specific regulation by antagonists and sodium ions. Mol. Pharmacol 37,383-394. 27. Brandt, D R. and Ross, E. M (1986) Catecholamme-stimulated GTPase cycle: Multiple sites of regulation by l3-adrenergic receptor and Mg*+ studied in reconstituted receptor-G, vesicles. J Biol Chem 261, 1656-1664 28. Sim, L. J., Selley, D. E., and Childers, S. R. (1995) In vitro autoradiography of receptor-activated G proteins m rat brain by agonist-stimulated guanylyl 5’-[y[35S]thio]-triphosphate binding. Proc. Natl. Acad Sci USA 92, 7242-7246. 29. Goodman, R. R., Snyder, S H., Kuhar, M J., and Young III, W. S (1980) Dlfferentiation of delta and mu opiate receptor locahzations by light microscopic autoradiography Proc Natl Acad Sci USA 77,6239-6243 30. Herkenham, M. and Pert, C. B. (1982) Light microscoptc localization of brain opiate receptors: a general autoradiographtc method which preserves tissue quality. J Neurosci 2, 1129-l 149. 3 1. Chu, D. C. M., Albin, R. L., Young, A. B., and Penney, J. B. (1990) Distribution and kinetics of GABAn binding sites in rat central nervous system’ a quantitative autoradiographic study. Neuroscience 34,341--357 32. Herkenham, M., Lynn, A. B., Johnson, M. R., Melvm, L. S., de Costa, B. R., and Rice, K. C. (1991) Characterization and localization of cannabinoid receptors in rat bram: a quantitative zn vitro autoradiographic study. J Neuroscz 11,563-583

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33. Jansen, E. M., Haycock, D. A., Ward, S. J., and Seybold, V. S. (1992) Distributron of cannabinoid receptors m rat brain determined with ammoalkylindoles. Brain Res. 575,93-102.

34. Sim, L. J., Xiao, R., and Childers, S. R. (1996) Identification of opioid receptorlike (ORLl) peptide-stimulated [35S]GTPyS binding in rat brain. NeuroReport 7,729-733. 35. Meunier, J.-C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., Butour, J.-L., Gmllemot, J.-C., Ferrara, P., Monsarrat, B., et al. (1995) Isolation and structure of the endogenous agonist of opioid receptor-like ORL, receptor. Nature 377,532-535.

36. Reinscheid, R. K., Nothacker, H.-P., Bourson, A., Ardati, A., Henningsen, R. A., Bunzow, J. R., Grandy, D. K., Langen, H , Monsma, F. J., and Civelli, 0. (1995) Orphanin FQ: a neuropeptide that activates an opiord-like G protein-coupled receptor. Sczence 270,792-794. 37. Sternweis, P. C. and Robishaw, J. D. (1984) Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. J. BIOI Chem 259, 13,80&13,813. 38. Wieland, T. and Jakobs, K. H. (1994) Measurement of receptor-stimulated guanosme 5’-0-(y-thro)triphosphate binding by G-proteins. Methods Enzymol. 237,3-13. 39. Sim, L. J., Selley, D. E., Dworkin, S. I., and Childers, S. R. (1996) Effects of chronic morphine administration on mu opioid receptor-stimulated [35S]GTPyS autoradiography in rat brain. J. Neurosci. 16,2684-2692. 40. Srm, L. J., Hampson, R. E., Deadwyler, S. A., and Childers, S R. (1996) Effects of chronic treatment with A9-tetrahydrocannabinol on cannabinoid-stimulated [35S]GTPyS autoradiography in rat brain. J. Neurosci. 16, 8057-8066.

Agonist-Induced High-Affinity GTP Hydrolysis as an Index of Receptor-Mediated G Protein Activation in Mammalian Brain Membranes Yuji Odagaki and Kjell Fuxe 1. Introduction Heterotrimeric guanine nucleotide-binding regulatory (G) proteins, composed of a-, p-, and y-subunits, play pivotal roles in many receptor-mediated transmembrane signaling processes. Although detailed regulatory modes of G protein-mediated signal transduction have not yet been fully elucidated, the following scheme is generally accepted (Fig. 1). In an inactive state, G protein exists as an associated form of GDP-bound a-subunit and fiy-subunits (Fig. IA). When an agonist is bound to the cell-surface receptor, the receptor is conformationally changed and interacts with the G protein so that GDP bound to the a-subunit is released and replaced with GTP (Fig. 1B). This GDP-GTP exchange reaction turns on the G protein activation process, in which GTPbound a-subunit is dissociated from py-subunits (Fig. lC), resulting in a modulation of the function of effector molecules such as adenylyl cyclase, phospholipases, and cGMP phosphodiesterase (Fig. 1D). The a-subunit of G protein has inherent high-affinity or low-K, GTPase activity, which hydrolyzes GTP on the a-subunit into GDP and inorganic phosphate (P,) (Fig. 1E). This turn-off reaction results in reassociation of GDP-bound a-subunit and &-subunits to end the activation cycle of G protein (Fig. IA). Such properties of the receptor-mediated G protein activation/inactivation cycle allow us to use several biochemical techniques to investigate the function of G proteins that are coupled with, and activated by, the receptors. These include receptormediated GDP release, GTP binding, and GTP hydrolysis. In 1988, Avissar et al. reported that isoproterenol- and carbachol-stimulated [3H]GTP binding in rat cerebral cortical membranes was abolished by the From

Methods In Molecular Biology, vol 83’ Receptor Srgnal Transductron Edlteci by R A J Chalks Humana Press Inc , Totowa, NJ

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Protocols

Odagaki and Fuxe

Fig. 1. Schematic diagram of receptor-mediated activation/mactivation cycle of G protein (see Introduction for detailed comments). A, agonist; R, receptor protem; E, effector molecule; a, a-subumt of G protein; Pr, &-subunits of G protein; S, substrate, P, product.

antimanic drug lithium m vttro as well as ex vivo (I). This report and their following work focused the attention of psychopharmacologists and biological psychtatrists on the relevance of G proteins or receptor-G protein couplmg as potential target sites of action of many psychotropic drugs, and even as possible contributors to the pathogenesis of some psychiatric disorders (for reviews, see refs. 2 and 3). One of the authors (Odagaki) tried to replicate their results, but failed to detect reliable effects of agonists on [3H]GTP binding sites in rat brain membranes. Today, agonist-induced GTP binding has been performed generally by using [35S]GTPyS as a radiohgand even m the native membrane system, and the methods are described in detail in a separate chapter of this monograph. The failure to detect agonist-induced [3H]GTP binding

Agonist-Induced GTP Hydrolysis

135

in brain membranes turned our focus to the development of a method for agomst-induced GTP hydrolysis as a way to probe receptor-G protein coupling in native brain membranes. Since Cassel and Selinger first described catecholamine-stimulated GTPase activity in turkey erythrocyte membranes m 1976 (4), there have been more than 200 reports in which agonist-stimulated high-affinity GTPase activity was utilized for many different kinds of receptors and the receptor-coupled G proteins. With the exception of some peripheral tissues, including human platelet membranes, this method has been utilized mainly in reconstitution systems and m cultured cell membranes, probably because of the simplicity of detection of agonist-stimulated activity and the ease with which such results can be interpreted. In contrast, native membrane preparations have very high background nonspecific GTP hydrolyzing activity, making the signal-to-noise ratio too small to be definitely and reliably detected, compared to simpler reconstituted and cultured cell systems.With regard to membranes prepared from mammalian brain, muscarinic acetylcholine receptor- and opioid receptor-mediated high-affinity GTPase activities have been most frequently reported (Table 1). Even in these cases, however, the signal-to-noise ratios do not seem high enough, which implies that other receptor-mediated high-affinity GTP hydrolyzing activities should be assayed with better care and technology to mmimize basal background activities and/or variability of the values. There have been some articles that describe the assay itself, especially from a practical point of view (5,6). Here, we describe in detail our own method for GTP hydrolysis that can be applied to an investigation of several receptor-coupled G proteins and their functional activation in native brain membranes.

2. Materials 1 [Y-~~P]GTP (30 Ci/rmnol) is purchasedfrom DuPont/New EnglandNuclear (Boston, MA) On arrival, it should be divided into several small portions and stored at -70 to -80°C to avoid frequent thawing and freezing. 2. All otherreagentsnecessaryfor theassayareobtainablefrom Sigma(St.Louts,MO). 3. Polypropylene microcentrifuge tubesat 1.5 mL are useful astest tubes in which the enzymereaction 1sallowed to occur.For a smoothandswift handling, lids of the tubes should be cut off beforehand. The use of a repetitive pipeter (Multipette@;Eppendorf, Hamburg, Germany) is recommended,as correct and quick pipeting is the key to minimal mtra-assayvariation. 4. TED buffer: 5 mM Tris-HCl, 1 mM EDTA, 1 mA4dithiothreitol, pH 7.4 and 50 mMTris-HCl buffer (pH 7.4) for membranepreparation: Storeat 4’C. Sucrose (lo%, w/v) should be dissolved in TED buffer on the day of membranepreparation (TED/sucrosebuffer). 5. The following solutions necessaryfor preparing the GTPaseassaymixture can be stockedin a refrigerator: 1M Tris-HCl, pH 7.4; 1M MgC12;10 mM adenyly-

Table 1 Receptor-Mediated High-Affinity in Mammalian Brain Membranes

GTPase

Number of references (examples)

Receptors Acetylcholine Muscarinic acetylcholine

Activity

19 [Onali et al. (1983) Mol. Pharmacof Ghodsi-Hovsepian et al. (1990)

24,380,

Blochem. Pharmacol. 39,1385]

Opiate p-Opioid 6-Opioid

6 (14)

5 [Georgoussi and Zioudroun (1993) Bzochem. Pharmacol

tc-Opioid Unspecified Adrenaline at-Adrenoceptor

45,2405]

3 [Clark et al. (1986) Life Sci. 39, 1721; Ueda et al.b (1987) Eur. J. Pharmacol 138,129] 10 [Franklin and Hoss (1984) J Neurochem. 43, 11321 2 [Villalobos-Molma

et al.” (1992) Bram Rex

590,303]

Dopamine Dopamine Dt-like

1 [Tirone et al. (1985) J. Cyclic Nucl. Prot. Phosph Res 10,327]

Dopamine D,-like

6 [Onali and Olianas (1987) Biochem. Pharmacol. 36,2839; (12)]

Other dopamine receptor S-Hydroxytryptamine (5HT) 5-HT,, y-Aminobutyric acid (GABA) GABAn Adenosine Adenosine A,

1 [Yue et al. (1994) Life Sci. 54, PL413] 3 (13) 7 [Odagakt and Fuxe (1995) Bruin Res. 689,129]

4 [(II);

Sweeney and Dolphin’

J. Neurochem

Glutamate Group-II mGluR Endothelm (ET) '=B

Miscellaneous Somatostatin

(1995)

64,2034]

1 (s)

1 [Sokolovsky (1993) Cell. Signal. 5,473] 1 [Eva and Costa (1987) .I Pharmacol

Exp. Ther

242,888]

Kyotorphin Cannabinoid

1 [Ueda et al. (1989) J. Biof Chem 264,3732] 2 [Pacheco et al. (1993) Brain Res. 603, 1021

“Antagonists have negatwe mtnnslc actwtles. bActivation of K-opioid receptor leads to inhlbitlon of high-affinity GTPase. CProbably interpreted incorrectly. See ref 15 for detailed discussion. dAdenosine A2 receptor may be also partially mvolved in 5’-(N-ethylcarboxamlde)adenosmestimulated high-affinity GTPase

Agonist-Induced GTP Hydrolysis

737

limidodiphosphate; BSA (10 mg/mL) (at most for several weeks); 10 WEDTA; 10 n&f EGTA (dissolved in 50 nuI4 Trismaa’ base); 10 miM drthiothreitol; and 1M NaCl. 6. Other solutions are prepared fresh on the experimental day: 10 mM ATP, 100 mM phosphocreatine, creatine phosphokinase (1000 UimL dissolved in 10 mg/mL BSA solution), 10 m&I cyclic AMP, 10 nuI4 3-isobutyl- 1-methylxanthine, and 50 or 100 @GTP. 3-Isobutyl- 1-methylxanthme is soluble in hot distilled water at a concentration of 10 n&f. Appropriate concentration of GTP solutron (usually 400 @4, to yield the final concentratron of 100 ClM; see Section 3 2., step 3) should also be prepared to define the nonspecific, low-affinity GTP hydrolyzing activity. 7. Activated charcoal (Norit A; Sigma, St. Louis, MO) suspension (5%, w/v): Phosphoric acid solutron (20 mM, pH 2.5) can be kept in a refrigerator Make 5% (w/v) suspension of activated charcoal (Norit A) in a capped conical polypropylene tube.

3. Methods 3.7. Membrane

Preparation

Rat brain membranes are prepared in the following way, which is a slight modification of the method of Ravindra and Aronstam (7). 1. Male Sprague-Dawley rats weighing 200-250 g are killed by decapitation and their brains are quickly removed. The cerebral cortrces, hippocampr, and striata are dissected on ice and homogenized in 5 mL of ice-cold TED/sucrose buffer (see Section 2., item 4), using a motor-driven Teflon/glass tissue grmder (20 strokes) (see Note 1) 2. All centrifuge procedures should be made at 0-4”C. Each homogenate is centrifuged at 1OOOgfor 10 mm. The resulting supematant is decanted to another centrifuge tube and kept on ice; the pellet is vortexed in 5 mL of TED/sucrose buffer, followed by another centrimgation at IOOOg for 10 min. 3. The combined supernatant is centrifuged at 9OOOg for 20 min, and the pellet is resuspended in 10 mL of TED buffer and centrifuged again at 9OOOg for 20 min. 4. The pellet is resuspended in 10 mL of TED buffer and kept on ice for 30 min. The suspension is finally centrifuged at 35,000g for 10 mm, resulting in the pellet that is then vigorously vortexed in an appropriate volume (3.0 mL for cerebral cortex, 1.5 mL for hippocampus, and 1.O mL for striatum) of 50 mA4 Tns-HCl buffer (pH 7.4) to produce the homogenate, the protein concentration of which should be within a range of 1.6-3.2 mg/mL. 5. This homogenate is divided into lSO+L aliquots in plastic tubes, which are then frozen quickly on fine-grained dry ice (see Note 2). A residual portron IS available for determination of the protein concentratron. 6. Frozen aliquots of the homogenate can be preserved at -70 to -80°C for at least 3-4 mo without any deterioration in activity.

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Odagaki and Fuxe

3.2. GTPase Assay 1. On the expenmental day, a tube containing the homogenate is taken from the deep freezer and inserted m compact powdered ice to allow the homogenate to thaw slowly (see Note 2). It takes the homogenate more than 2 h to thaw, during which time a mmimally necessary volume of the assay mixture (50 pL/assay) can be prepared (see Note 3). 2. The assay mixture should be prepared by mixing the solutions listed in Section 2., items 5 and 6, so that the followmg constituents are contained m a final reaction mixture (100 pL) at the indicated final concentrations (see Note 4). 50 mM Tris-HCl, pH 7.4,O 3 fl GTP ( [Y-~~P]GTP plus unlabeled GTP), 2 n-& MgCl,, 0 5 mMATP, 0.5 mMadenylyhmidodiphosphate, 5 tiphosphocreatine, creatine phosphokmase (5 U/assay), BSA (50 pg/assay), 0 1 mM EDTA, 0 2 mM EGTA, 0.2 mM dithiothreitol, 0.5 mM cyclic AMP, 0 5 mM 3-isobutyl-1-methylxanthine, and 100 mM NaCI. Because the half-life of 32P is relatively short (T,,, = 14.3 d), the decay of the radioactivity should be considered Usually, either of the following two conditions is selected according to the decay to yield 0 3-l .O x lo6 cpmassay: 0.1 pJ4 [Y-~~P]GTP plus 0.2 pA4GTP or 0.2 pA4 [Y-~~P]GTP plus 0.1 M GTP. [Y-~~P]GTP should be added last and just before starting the enzyme reaction to avoid unnecessary exposure to the radioactivity. 3. The thawed brain membranes (150 l.tL) are diluted with 1350 pL of 50 rr&! TrisHCl buffer (pH 7.4), and using a repetitive prpeter, 25-pL aliquots (4-8 pg protein/assay) are put into 1.5-n& microcentrifuge tubes placed in ice-cold water. Another 25 pL of drstilled water containing an appropriate concentration of compound(s) (agonist and/or antagonist) or unlabeled GTP (final concentration 100 @4, to define the low-affinity GTP hydrolyzing activity) is added to each tube. Finally, the above-mentroned assay mixture containing [Y-~~P]GTP IS prpeted by 50 pL per assay tube with a repetitive pipeter to make a final reaction mixture of 100 p.L (see Note 5). 4. The tubes are vortexed rapidly and immersed in a 30°C water bath. After the incubation of the tubes at 30°C for an appropriate time (usually 15 min in our laboratory, see Note 6), the tubes are transferred to an ice-cold water bath, followed by the addition of 500 @., of ice-cold 20 mM phosphoric acid (pH 2 5) containing 5% (w/v) activated charcoal (Norit A) (see Section 2., item 7) by a repetitive pipeter (see Note 7). 5. The tubes are kept chilled for approx 30 min and centrifuged at 1 l,OOOg for 10 min (see Note 8). An aliquot (200 pL) from the supernatant fraction of each tube is put into a scmtillation vial, to which 5 mL of scintillation cocktarl is added The vial is shaken and the radioactrvity is determined by a liquid scmtillation spectrometer with a counting time set for at least 5 mm (see Note 9). Shown in Fig. 2 is the result of one experiment m which metabotropic glutamate receptor (mGluR)-mediated G protein GTPase is activated by L-glutamate in rat cerebral cortical membranes (via Group-II mGluRs; see ref 8). It can be recognized that intra-assay variation of duplicate determmations 1s

Agonist-Induced

GTP Hydrolysis

139

Log [Glutamate] (M)

Fig. 2. MIilutamate-stimulated GTP hydrolysis in rat cerebral cortical membranes in a representative experiment. The raw data of duplicate determinations measured in cpm are presented. [Y-~~P]GTP addedin eachassaytube is 833562 cpm.Seeref. 8 for the properties of this response.

minimal. Replicate cpms are averaged, and the low-afftnity and agonist-insensitive GTP hydrolyzing activity (determined in the presence of 100 @4 unlabeled GTP) is subtracted from all other values to define the high-affinity GTPase activity.

4. Notes The most critical point of this method seems to be to minimize the variability of assaysto overcome the huge background GTP hydrolyzing activity seen in the native brain membranes. However, when a signal-to-noise ratio is still too small, even under condition of low variability for the assay,further changes may be necessary. For example, the use of synaptic plasma membranes instead of crude membranes can be considered to reduce the background GTP hydrolysis activity. Alternatively, some fatty acids, such as cis-vaccenic acid (9,ZO) may be useful to facilitate the interaction between receptors and the relevant G protein molecules. 1. As in an assay for adenylyl cyclase activity, it appears better to avoid using sonication or a Polytron-type homogenizer to obtain the homogenate. 2. The membranes should be frozen as quickly, and thawed as slowly, as possible. 3. For 40 assays, for instance, prepare 2500 & of the assay mixture. 4. The assay conditions can be modified appropriately, according to the purpose of the experiment or to the special situation of the target receptor-mediated signal-

140

5. 6.

7.

8. 9.

Odagaki and Fuxe mg. For mstance, adenosine At receptor-coupled GTP hydrolysis was measured m a modified reaction mixture, m which 0.2 U/assay of adenosine deaminase was added and 3-isobutyl- 1-methylxanthine was omitted (I I). The assay mixture should also be put directly mto a scintillation vial in order to quantify the total [Y-~~P]GTP (in cpm) added to each assay tube in the experiment Under our assay condittons, the time-course of high-affinity GTPase activity is curvilinear (12,13). The standard incubation time should be decided in each laboratory, so that the hydrolysis of [Y-~~P]GTP does not exceed 20-30% of the total [Y-~~P]GTP added to each assay tube. Ueda et al. (14) recommends washing the charcoal before use to reduce variability. From our own experience, however, the most important point at this stage of the procedure seems to be rapid and accurate pipeting of the charcoal suspension. Therefore, homogenous charcoal suspension should be maintained by vigorous vortex-mixing and shakmg, immediately followed by rapid plpeting (wlthm 1&I 5 s for 20 assays). The tip of a Combitip@’ should be cut off before use to avoid clogging. It does not seem that the precise centrifugation condttions are important. Thus, almost any bench centrifuge can be used. In order to minimize measurement variability, set a counting time (5 or 10 min) for each sample, giving a 298% confidence m the cpm value obtained.

Acknowledgments We would like to thank Satoko Takabayashi for her help in bibliographic research. The work in the Department of Neuroscience, Karolinska Institute, was supported by grants from the Karolinska Institute, the Swedish Medical Research Council (04X-7 15), and Knut and Alice Wallenberg Foundation.

References 1. Avissar, S., Schreiber, G., Danon, A., and Belmaker, R. H (1988) Lithium inhibits adrenergic and cholinergic increases in GTP binding in rat cortex. Nature 331, 440-442. 2. Avissar, S. and Schreiber, G. (1992) The involvement of guanine nucleotide binding proteins m the pathogenesis and treatment of affective disorders. Biol. Psychzat. 31,435-459. 3. Manji, H. K. (1992) G proteins: implications for psychiatry. Am J. Psychzat. 149, 746-760. 4 Cassel, D. and Selinger, Z. (1976) Catecholamine-stimulated

GTPase activity in turkey erythrocyte membranes. Biochlm. Biophys. Acta 452,538-55 1. 5. Gierschik, P., Bouillon, T., and Jakobs, K. H. (1994) Receptor-stimulated hydrolysis of guanosine 5’-triphosphate in membrane preparations, in Methods in Enzymology, vol. 237 (Iyengar, R., ed.), Academic, San Dtego, CA, pp. 13-26 6. Sweeney, M. (1995) Measurement of the GTPase activity of signal-transducing G-proteins in neuronal membranes, m Methods in Molecular Biology, vol. 41 (Kendall, D. A. and Hill, S. J., eds.), Humana, Totowa, NJ, pp. 5 l-6 1.

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7. Ravmdra, R. and Aronstam, R S. (1990) Influence of anti-tubulin antibodies on muscarinic receptor modulatron of G protein GTPase activity in rat striatum. Biochem. Pharmacol. 40,457-463. 8. Odagakr, Y., Nishi, N , and Koyama, T. (1996) Functional coupling between metabotropic glutamate receptors and G proteins m rat brain membranes. Eur. J. Pharmacoi 300,15 l-l 54. 9. Orly, J. and Schramm, M. (1975) Fatty acids as modulators of membrane functions: catecholamine-activated adenylate cyclase of the turkey erythrocyte Proc Natl. Acad. SCI. USA 72,3433-3437. 10 Briggs, M. M. and Letkowitz, R. J. (1980) Parallel modulation of catecholamine activation of adenylate cyclase and formation of the high-affinity agonist * receptor complex in turkey erythrocyte membranes by temperature and cis-vaccemc acid. Blochemwtry 19,446 l-4466. 11. Odagaki, Y. and Fuxe, K. (1995) Functional coupling between At adenosine receptors and G-proteins m rat hippocampal membranes assessedby high-affinity GTPase activity. Br. J Pharmacol. 116,2691-2697. 12. Odagaki, Y and Fuxe, K. (1995) Functional couplmg of dopamine D, and muscarmic cholinergic receptors to their respective G proteins assessed by agonistinduced activation of high-afflmty GTPase activity m rat striatal membranes. Biochem Pharmacol 50,325-335 13. Odagaki, Y and Fuxe, K. (1995) Pharmacological characterization of the 5hydroxytryptamine-1A receptor-mediated activation of high-affinity GTP hydrolysis m rat hippocampal membranes. J. Pharmacol. Exp Ther 274,337-344. 14. Ueda, H., Misawa, H., Katada, T., Ui, M., Takagr, H., and Satoh, M. (1990) Functional reconstitution of purified G, and G, with p-opioid receptors in guinea pig strratal membranes pretreated with micromolar concentrations of N-ethylmaleimtde. J Neurochem. 54,841-X48. 15. Odagaki, Y., Dasgupta, S., and Fuxe, K. (1995) Additivrty and non-additivity between dopamine-, norepinephrine-, carbachol- and GABA-stimulated GTPase activity. Eur. J PharmacoL-Mol. Pharmacol. Set 291,245-253.

11 Use of Random-Saturation Mutagenesis to Study Receptor-G Protein Coupling Ethan S. Burstein, Tracy A. Spalding, and Mark R. Brann 1. Introduction The advent of powerful molecular techniques to study protein structure and function allows the modification of the codmg region of genes to produce proteins with altered structures that can be studied for changes in biochemical properties. The most common way to do structure/function studies is by site-directed mutagenesis, m which a particular residue in a protein is selected and systematically altered. Because of the labor-intensive nature of this approach, it is necessary to limit the scope of such studies to relatively few residues of a protein. Predictions of which residues are of functional importance are usually based solely on homology to related proteins and are unreliable, because sequence homology is a reflection of evolutionary relatedness as well as function. Also, the high degree of sequence homology present in many gene families makes it difficult to limit the scope of such studies. Another disadvantage of site-directed approaches is that often mutations introduced at critical residues destroy function, which can be caused by many trivial effects unrelated to the question bemg tested, such as improper protein folding, and so on. In contrast, random-saturation mutagenesis does not require one to presume the functional importance of a particular residue a priori, since thousands of amino acid substitutions are tested with no bias regarding the type and location of substitutions made. This overcomes the need to infer structural significance from sequence comparisons, since all amino acids in a given region are subjected to mutations (Id). Using an assay based on retention rather than disruption of function, one can identify which amino acid substitutions are functionally allowed, the likelihood of misinterpretation inherent in evaluation From

Methods II) Molecular Biology, vol 83 Receptor Signal Transduction EdIted by R A J Chalks Humana Press Inc , Totowa, NJ

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Protocols

Burstein, Spalding, and Brann

144

of negative data is significantly reduced, and activated phenotypes, or gain of function mutations, are particularly easy to identify (6). When a domain is saturated with mutations, one can make inferences about amino acid substitutions that are never recovered as functional proteins (1-s). A broad range of allowed amino acid substitutions can be identified, from which patterns can be inferred and critical mutation-intolerant residues singled out for further study (7). The ability to establish structure-function relationships using randomsaturation mutagenesis is limited by the capacity to functionally screen large numbers of receptors. We have developed a reliable and facile screen based on the observation that many receptors and oncogene products induce proliferation/transformation responses in NIH-3T3 cells (8). These stimulatory effects can be monitored using a reporter gene assay (4,9,10). In our assay, ligands select and amplify cells that express functional receptors (R-SAT, for Receptor Selection and Amplification Technology, patents pending). This assay allows graded responses to be measured, permitting a quantitative evaluation of mutant receptor phenotypes. As shown in Fig. 1, there is a time- and dose-dependent amplification of cells transfected with the human m5 muscarinic acetylcholine receptor and P-galactosidase,asmeasured by the absorbance of a P-galactosidase substrate. Extensive work has demonstrated that the pharmacology of ligands at receptors, evaluated using R-SAT, accurately reflects results obtained with traditional assays (9). Thus, functional receptors can be cloned from a library of mutants by virtue of their ability to induce ligand dependent (or independent, see ref. 6) proliferative responses. We have employed R-SAT in combination with random-saturation mutagenesis to investigate the structure-function relationships of muscarinic receptorG protein coupling (4-7). Figure 2 shows examples of sequencesderived from a library of receptors containing mutations randomly mtroduced into a 18-amino acid segment. Inspection of sequences derived from receptors randomly selected from the library reveals that there is a random distribution of mutations. However, the pattern of functionally tolerated mutations is nonrandom, with several residues that tolerate only conservative or no mutations. These residues define a functional consensus sequence, which was then studied using site-directed mutagenesis (7). Thus, random saturation mutagenesis, combined with a reliable functional screen, is a useful method to obtain structure/mnction relationships of receptors (and other proteins). 2. Materials

2.1. Mutagenesis/Library

Construction

1 Oligonucleotides were synthesizedon a Model 391 DNA Synthesizer(A.B.I. [Columbia, MD]) or purchasedfrom commercial vendors.

Random-Saturation Mutagenesis

145

1.0 II-

0.8

0.6 / -

0.4

0.2 1. 0 0

2

4

6

8

10-g

10-g

1oJ Carbachol

Day

10-6

10-5

1o-4

(M)

Fig. 1. R-SAT assay. (A) Time-course. m5 and @galactosidase cDNA’s were cotransfected into NIH-3T3 cells and induced P-galactosidase activity assayed at the indicated times, as described m Sections 2. and 3. The responses were calculated from the baseline and maximum responses derived from least-squares fits of full dose/ responses at each time-point (filled triangles, maximal response; open circles, basal response). (B) Carbachol dose-response of the P-galactoadase amplification on d 5. Plotted is absorbance at 405 nm (filled triangles) vs carbachol concentration.

2. Pfu DNA polymerase (Stratagene, La Jolla, CA). 3. Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, and T4 DNA polymerase (New England Biolabs [Beverly, MA]). 4. Seaplaque low-melting agarose (FMC Bioproducts). 5. Library efficiency competent E. coli (DHScl strain) (Gibco-BRL [Gaithersburg,

MW.

6. Luria Broth or agar (Gibco BRL) with ampicillin, recipes).

2.2. R-SAT/Functional

SOC medium (see ref. 16 for

Screen

1. NIH-3T3 cells (ATCC CRL 1658). 2. o-nitrophenyl-/3-u-galactopyranoside and nonidet P-40 (Sigma [St. Louis, MO]). 3. Dulbecco’s modified Eagles medium (A.B.I.) supplemented with 4500 mg/L glucose, 4 nM L-glutamine, 50 U/mL penicillin G, 50 U/n& streptomycin, and 10% calf serum (Sigma) or 2% cyto-SF3 synthetic supplement (Kemp Laboratories).

146

Burstein, Spalding, and Brann A

Introduced

mutations are randomly distributed

212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 221228

Hm5 Introduced Substitutions

LYCRIYRETEKRTKDLAD HSSNSHDKKGMGRTGPVH HCGLSNNVIKGHSKYPGC R F L NNLV PFI VN D

F

B

229

Functionally

M

VNHS N N D

tolerated substitutions

V N A

A

PV TD

are non-random

212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 22.8 229

Hm5 Consensus Tolerated

Mutations

LYCRIYRETEKRTKDLAD

L Y -

- I Y -

VFGLM

y

GQM S

I

R

F

:: W F

L

- T -

WVAVE LVAVE GLAVT QAAAT ii N D D

- R -

- -

-

- -

NTVNDV NTVPRG LQNRGA IMNRPV ‘D; D

E

;

: E

Fig. 2. Substitution patterns in randomly mutated receptors. The m5 muscarinic receptor was saturated with mutations m the N-terminus of the third mtracellular loop (N-i3) region, including amino acid residues 212-229, as described in Section 3. Individual clones were sequenced, and the observed substitutions compiled into lists according to amino acid position. (A) Substituttons comptled from 14 receptors selected randomly from a library of -1000 mutant receptors and sequenced. (B) Substitutions compiled from 37 fully functional (E&c c 5 x EC&vT and maximum response >0.8 x maximum responsewT) receptors cloned, using R-SAT from the same library of -1000 receptors. The consensus is composed of residues m which no radical substitutions were tolerated.

4. 96-well, 24-well, 6-well, and lo-cm tissue culture dishes (Falcon [Lincoln Park, NJ]). 5 Hanks balanced salt solution with and without magnesium chloride, magnesium sulfate, and calcium chloride, Trypsin-EDTA (all from Gibco-BRL)

Random-Saturation

147

Mutagenesis B

A

Apa1

Xbal

0

Ligale ml0 @D-m5

0

Transkxm E. Coli

Fig. 3. Mutagenesis of the C-terminus of the third intracellular loop (C-i3) of m5 (taken from ref. 4). (A) Region to be mutagenized. Wild-type sequence is indicated. Abbreviations: TM’s l-7 are transmembrane regions 1-7. il, i2, and i3 represent intracellular loops 1,2, or 3. (B) Library construction strategy. PCR was performed using primers pl-p4. The p2 primer was comprised of residues 423-444. The outer primers (Pl and P4) contain Apa1 and XbaI restriction sites for subsequent cloning. The two PCR products were treated with T4 DNA polymerase to create blunt ends, ligated to yield concatamers, and restricted with ApaI and XbaI to release the randomly mutated (*) Ci3*ApaI-XbaI inserts. Inserts were ligated into a ApaIIXbaI fragment of the pcD-m5 yielding a population of mutant m5 receptor DNA @CDmS-Ci3*).

3. Methods 3.1. Oligonucieotides 1. Oligos used were synthesized on a Model 391 DNA Synthesizer (A.B.I.) and gelpurified or were purchased from commercial vendors. 2. To incorporate mutations, an equimolar mixture of the four bases were substituted at a 15% rate (11.25% misincorporations) for wild-type nucleotides during synthesis of the mutagenic primer P2 (see Fig. 3). P2 spans the entire segment to be mutated. The primers Pi and P4 contain restriction sites for subcloning. P2 was phosphorylated with polynucleotide kinase prior to PCR amplification.

3.2. PCR Reactions 1. Two PCR products were prepared (Pl-P2 and P3-P4) for library construction (see Fig. 3) on a Techne PHC-3 Dry-Block Thermocycler, using the GeneAmp PCR kit (Perkin-Elmer Cetus).

148

But-stein, Spalding, and Brann

2 Cycling conditions included an initial 5 min melting step at 94Y!, followed by 30 cycles of 94“C (1 min), 45OC (1 min), with a 2 min ramp to 72°C (2 min) and a final 10 min extension at 72’C. Lower stringency annealing conditions were used, based on the expectation of multiple mismatches between mutated primers and templates.

3.3. Cloning/Construction

of DNA Libraries

1. PCR products were treated with T4 DNA polymerase (3 U/100 pL) to create blunt ends, ethanol precipitated, resuspended in one-fifth the original PCR volume, ligated to create concatamers, and digested with suitable restriction enzymes to release the randomly mutated inserts. 2. Inserts were ligated into a fragment of the receptor-expression vector containing compatible ends to yield a population of mutant receptor DNA. 3. Libraries of recombinant mutant receptors were prepared by transformmg competent E. cok. Plasmid DNA was punfled from mdrvidual clones or pools of clones using QIAWell from Qiagen (Chatsworth, CA) according to the manufacturers instructions and used for sequencing and functional assays.

3.4. Cell Culture NIH-3T3 cells were incubated at 37°C in a humidified atmosphere (5% CO*) in Dulbecco’s modified Eagle’s medium supplemented with 4500 mg/L glucose, 4 nkf L-glutamine, 50 UlmL penicillin G, 50 U/mL streptomycin (A.B.I.), and 10% calf serum (Sigma). 3.5. Transfection Procedure and Functional R-SAT assayswere performed as follows:

Assays

1. Cells were plated 1 d before transfection using 1 x 1O6cells in 10 mL of media per IO-cm plate, 2 x lo5 cells in 2 mL of media per 6-well plate, or 7.5 x 104 cells in 0.5 mL of media per well of a 24-well rack. 2. Cells were transfected by calcium precipitatton as described by Wigler et al. (II), using 2.5 ~18of receptor, 2.5 pg PSI-@galactosidase (Promega, Madrson, WI), and 10 pg of salmon sperm DNA (Srgma) per IO-cm plate and proporttonately more or less for bigger or smaller dishes, respectively. 3. One day after transfection media was changed. Two days after transfection, cells were trypsinized and aliquoted into the wells of a 96-well plate (100 @,/well). One lo-cm plate yields enough cells for 96 wells. 4. Cells were combined with ligands in DMEM supplemented wrth 2% cytoSF3 synthetic supplement (Kemp Laboratories), instead of calf serum, to a final volume of 200 &/well. For the 24-well format, the trypsinization step was omitted (see Note 7). 5. After 5 d in culture, l3-galactosidase levels were measured essentially as described (12). The media were aspirated from the wells and the cells rinsed with phosphate-buffered saline (PBS), pH 7.4. 200 pL of PBS, with 3.5 mA4 o-nitrophenyl-P-o-galactopyranoside and 0.5% nonidet P-40 (both Sigma), was added

Random-Saturation Mutagenesis

149

to each well and the 96-well plates were incubated at room temperature. After 16 h the plates were read at 420 run on a plate-reader (Bio-Tek EL 3 10 or Molecular Devices). 6. Dose-response data from R-SAT assays were fit to the equation: R=A+(B-A)(x)/(c+x) where A = minimum response, B = maximum response, and c = EC,, (R = response, x = concentration of ligand). Maximum response values are normalized according to an internal standard based on the fact that NIH-3T3 cells endogenously express prostanoid FP receptors. Thus, two wells of a dose response are exposed to the prostanoid agonist cloprostanol(lO0 nM, EC5a = 1 nM, Cayman Chemicals [Ann Arbor, MI]), combined with a suitable antagonist to suppress any constitutive activity of the transfected receptors.

4. Notes 1. Mutagenesis: Mutations are introduced during synthesis of the oligonucleotide to allow quantitative control of the rate of base misincorporation, and ensure a random distribution of mutations, unlike in vivo and chemical methods of mutagenesis that are subject to sequence specific bias {13,14). The following equation predicts the expected probability of mutations: P(x) = [N!/x!(N

- x)!]CX( 1 - C)N-x

where P(x) is the probability of x mutations per oligonucleotide, N is the length of the oligonucleotide, and C is the doping rate (25). To maximize the information per clone, it IS desirable that a high proportion of the clones contain multiple mutations. Too low a rate would result in a high frequency of wild-type receptors that would provide no information, but too high a rate would result in a very low proportion of active receptors. As shown in Table 1, for a 60-base oligonucleotide at a mutation rate of 2% (doping rate of 2.67% equimolar mix), on average 29.8% of the receptors will have no base changes. A mutation rate of 11.25% (doping rate of 15%) would be predicted to give ~1% clones with no mutations, and -80% clones with 3-8 mutations per clone, and a mutation rate of 20% will result in >80% clones, with more than 10 base changes per clone (see Table 1). Experimentally, using a doping rate of 15% on a 66-base oligonucleotide, we obtained an average of 10 base changes per receptor, resulting in 5 amino acid substitutions in receptors selected randomly from libraries. In contrast, we saw only 2.5-3.5 mutations per receptor in the cytoplasmic loops and 1.7 mutations per receptor in the transmembrane regions in functional receptors identified by R-SAT (4,5; Spalding et al., unpublished observation). A lower mutation rate in the functional receptors was expected, since the functional screen imposes selective pressure against mutations, ensuring that the observed mutation patterns in the functional clones is nonrandom (compare Fig. 2A,B). 2. Library representation and amino acid codon bias: Codon bias is an inherent problem when performmg random mutagenesis. It may require one, two, or three base

Burstein, Spalding, and Brann

150 Table 1 Frequency Distribution of Mutations Mutation rate Oligo length

0.02

0 11

60

60

Mutations/o 1igo 0

0.11 90

0.11 30

00 00

30 11.2 20 1 23.2 19.4 12.5 64 2.7 1.0 0.3 0.1

Frequent y, % 29.8 36.4 21 9 8.7 2.5 06 0.1 0.0

1 2 3 4 5 6 7 8 9

0.20 60

10 11 12 13 14 15 16 17 18 19

01 0.7 25 59 10.5 14.5 16.4 15.6 12.8 9.1 5.8 32 1.6 07 0.3

0.1

20

00 0.0 00 0.1 03 0.8 19 36 6.0 8.6

11.0 12 5 12.8 11 8 9.9 76 53 35 2.1 11 06

0.2 0.6 17 3.5 6.2 9.2

11 8 13 2 13.3 11.9 9.7 7.2 4.9 31 1.8 1.0 0.5 0.2 0.1

Frequency dtstrtbutions were calculated using the formula shown m Note 1 and using the indicated values for mutation rate, olrgonucleottde length, and expected mutation number per ohgonucleottde

changes to create a particular amino acid substitntton, depending on the starting and mutated codons To calculate the probability of a given amino acid substitntron occurring, one must first calculate the probability of any codon being changed to any other codon. Using a doping rate of 15%, the proportion of wild-type nucleotrdes will be 88.8% and the proportton of each other nucleotrde will be 3.8%. Therefore, starting with the codon GCT as an example, here are some probabilities: Condon GCT GAT GTA CGA

= (0.888)3 = (0.888)2 (0.038) = (0.888) (O.O38)2 = (0.038)3

Probabtlity 70% 3%

0.12% 0.005%

Random-Saturation Mutagenesis

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By setting up a matrix, one can do this for all possible codon changes. To determine the probability of getting a particular ammo acid substitution at a specific codon, sum the probabilities of that codon being changed to all codons for that amino acid substitution. Using Alanine (GCT) as an example, the chance of it changing to Aspartate (GAT or GAC) is [GCT to GAT = 3 00%] + [GCT to GAC = 0.12%] = 3.12%. Note that it is much harder to change Alanine (GCA) to Aspartate [GCA to GAT = 0 12%] + [GCA to GAC = 0 12%] = 0.24%, because two base changes are required. Thus, the bias inherent to this random mutagenesis protocol will depend on the specific sequence being mutated. The probability that a particular substitutton occurred in a given size library rs calculated as PN = [l - (1 - PJN], where P, is the probability of changing a codon to all codons for that ammo acid substrtution and N is the number of clones. For example, in 100 clones the chance of getting Ala (GCT) changed to Asp (GAT or GAC) is [ 1 - (1 - 0.03)100] or 95.3%, but changing Ala (GCA) to Asp is [ 1 - (l0.0024)roo] or 21.4%. One can easily extend this to amino acid class and calculate the chance of getting a basic, acidic, hydrophobic, and so on, substttution at a particular codon by summing the probabilities of changes to each residue within that amino acid class (Table 2) If one defines saturating mutagenesis as changing each residue at least once to each of the other major classes of amino acids, then the analysis in Table 2 indicates one can approach saturation in a library of 50(O) clones. Obviously, certain substitutions (e.g., to acidic residues) are less likely to occur. This problem can be overcome with an optimized doping scheme. Using a higher percentage of G’s at position one of each codon will favor acidic residues, although at the expense of other ammo acids. The strategy of using optimized dopmg schemes to favor subsets of amino acids is described more fully elsewhere (3). 3 Functional clones and mutation patterns: The size of the library required to achieve saturation is mathematically identical regardless of the tolerance of a given protein segment to mutation, but rt is necessary to screen larger libraries of a mutationally intolerant region to isolate enough functional clones to observe mutation patterns. The mutational tolerance of a domain will depend on the number of functionally critical residues and the variety of substitutions they will tolerate Interacting domains within a protein segment may further preclude observation of tolerated mutatrons (see Note 4). We have observed that the cytoplasmic loops of the m5 muscarimc receptor tolerate more mutations per receptor than the transmembrane regions (see Note 1). Similarly, the proportion of active receptors ranged from 2 to 10% for the cytoplasmrc loops (4,5), but 1% or less for the transmembrane domains (Spalding et al., unpublished observations). The low rate of positives m transmembrane domain libraries necessitated screening pools of clones (multiplexing; see Note 6). Alternatively, a slightly lower mutation rate could have been used to increase the proportion of active receptors m those libraries. As discussed above, because of codon bias, certain substituttons are improbable, making rt difficult to determine the functional importance of every residue with certainty unless one is willing to analyze very large libraries.

Table 2 Substitution AA GAY GUY GUY Ala Ala Ala Ser Ser Ser Ser Glu Glu Asp His LYS LYS MS f-b b3 fb 43 Met W TY

Rates bv Amino ClaSS

S S S S S S S S S S A A A B B B B B B B B H H H

Codon

GGG GGA GGTK GCG GCA GCT/C TCG TCA TCT/C AGTK GAG GAA GATK CAT/C AAG AGG AGA CGG CGA CGTK ATG TGG TAT/C

Acid Class Small, % 100 100 100 100 100 100 100 100 100 100 100 100 100 94 97 97 100 100 100 100 100 97 100 100

Hydrophobic, 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

%

Acids, %

Bases, %

Proline, %

100 100 100 100 100 100 51 51 51 51 100 100 100 100 100 100 51 51 51 51 51 51 51 100

100 100 100 87 87 77 87 87 77 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

51 51 51 100 100 100 100 100 100 51 51 51 51 100 51 51 51 51 100 100 100 51 51 51

Phe lle lle Val Val Val Leu Leu Leu Leu Lell

zi

ThlThr Thr Am CYS Gln Gln Pro Pro Pro

H H H H H H H H H H H N N N N N N N P P P

TTTK ATA ATT/C GTG GTA GTT/C TTG TTA CTG CTA CTTK ACG ACA ACT/C AATIC TGTIC CAG CAA CCG CCA CCTIC

100 97 100 100 100 100 100 100 89 89 94 100 100 100 100 100 89 89 100 100 100

100 100 100 100 100 100 100 100 100 100 100 99 100 100 100 100 100 100 100 100 100

51 51 51 100 100 100 51 51 51 51 51 51 51 51 100 51 100 100 51 51 51

77 100 98 87 87 77 87 87 100 100 100 100 100 98 100 100 100 100 100 100 100

51 51 51 51 51 51 51 51 100 100 100 100 100 100 51 51 100 100 100 100 100

Probablkes were calculated as described m Note 2, usmg a basis of a 500-clone hbrary and a mutation rate of 0.1125%. Codes for amino acid class are: S, small; A, acid, B, base, H, hydrophobic/large; N, neutral/medmm, P, proline. Codons for the same ammo acid that were C or T m the tird position were combined since there is no difference m theu mutation rates. Probabihhes are rounded off to the nearest percent

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Therefore, tt IS recommended that random mutagenesis be used to exclude MOST of the unmterestmg residues and identify a small subset of residues for further analysis For example, 12 of the 18 residues shown in Fig 2B (#214, 215, 218-222, and 224-229) tolerate radical mutations and can be excluded. The remaining important residues were subsequently analyzed by point mutations (7). 4. Interactions between mutations: Analyzing receptors that contam multtple mutations raises the possibihty of addittve effects of mutations. As a first approximation, most mutations can be treated individually Empirical data m which residues identified by random mutagenesis were then reanalyzed using site-directed mutagenesis supports this notion (4,7). It is difficult to gage the additive effects of multiple mutations Often the effect would be to further compromise a receptor that might tolerate the mutations mdividually, but It is also possible that mutations that would not be allowed individually might work together The chance of discovering mteracttve residues that rescue or increase function is a distinct advantage of random mutagenesis 5. Library design: When designing a random-saturation mutagenesis experiment, it IS preferable to restrict the analysis to small domains to keep the scope of the experiment manageable. Therefore, it is important to have some knowledge of where the important domains are before starting; otherwise one might waste effort mutating nonessentral parts of the protein (Note It IS important to distinguish random-saturation mutagenesis in which one wants to obtain mutation patterns from other random mutagenesis m which often an entire protein is mutated to produce a few clones with interesting phenotypes ) Segments of 20-25 residues can be conveniently spanned with a smgle obgonucleotide. This is sufficiently long to saturate an entire transmembrane domain of a receptor. Targeting longer segments increases the chance of mutating domains that serve different functions, such as hgand binding and G protein couplmg, mcreasmg the complexity of the libraries and the problems associated with mteractive mutations (see Note 4). Construction of mutagenized cassettes using blunt-end ligations allows one to place the mutations anywhere m the receptor; the identical protocol can be used to mutagenize adjacent domains, for example, a transmembrane segment and the adjoining cytoplasmtc loop This IS a particularly useful strategy for studying receptors, Alternatively, if there are convenient restrtctton sites avwlable tt IS not necessary to do blunt-end hgations, which are technically difficult Whenever posstble, one should introduce restriction sites durmg library constructton to distmguish recombinant receptors from religated wild-type receptors. In this manner one can ensure that there is no contamination by the parent plasmid, since even a low level of contamination will be over-represented in the positive clones identified in the functional screen 6. Production of library DNA: We have found that the preparation of the DNA is crucial to the success of the transfections and hence the functional screens, Qiagen prepared DNA on QIAwell or gravity flow Qiagen columns has been successful,

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spm columns has not. to ensure equal representation of each clone, but DNA can be prepared in pools when the proportion of active clones is expected to be less than 1%. A significant advantage of R-SAT IS the abihty to assay multiple receptors together. 7. Configuration of the functional assays: R-SAT can be configured in a variety of culture formats. Prehmmary screening of libraries is done in 24-well formats. In this format, cells are not trypsmized; rather, media is changed to media containing ligands. It is advisable to screen with an agonist, no drug, and an antagonist in order to identify constitutively activated receptors. Once identified, positive clones are subjected to a second round of analysis. In this case, larger scale transfections into &well, 10 cm, or even IS-cm dishes are trypsinized, split into 96-well dishes, and combined with full dilution series of ligands. This assures that cells at each dose of hgand were identically transfected. To control for the transfection efficiency of the reporter gene, one can use the fact that endogenous prostanoid receptors give a robust R-SAT response as an internal standard as described in Methods. We have found that this works well for assaying receptors, but is invalid for gene products that operate downstream of cell surface receptors. 8. Other consideration: NIH-3T3 cells that have a low passage number work best m R-SAT. Care must be taken not to disturb the monolayer of cells during media changes, especially in the 96-well format because tearing and loss of too many cells can reduce signal. Calf serum is required for propagation of the cells, recovery of the cells from transfection, and during the trypsimzation step to inactivate the trypsin. Use of either calf serum or the defined supplement Cyto-SF3 during incubation of cells with ligands will work, but Cyto-SF3 is recommended because there is less background, the likelihood of ligand degradation is reduced, and the possibility that endogenous ligands are present is eliminated. If cell survival during incubation with ligand is a problem, use a small amount of calf serum (0.5%) along with the defined supplement. 9. Applications/limitations: A stable ligand is a prerequisite. Most commercially available hgands give satisfactory results (6,9,10). It is advisable to test wild-type receptors for activity in R-SAT before embarking upon a random mutagenesis experiment. The background responses of endogenous receptors are generally low compared to the responses of transfected receptors, but this should be verified. R-SAT works best with G protein coupled receptors linked to phosphatidylinositide turnover, and with tyrosine kinase receptors (4,9,10) Heterotrimeric G proteins (18) and many nonreceptor protooncogenes are also active in R-SAT (Burstein et al., unpublished observations). G protemcoupled receptors linked to inhibition of adenylyl cyclase do not work as consistently (17) and m many cases must be coexpressed with a chimeric G protein between Go?, and the last five ammo acid residues of Ga,z to see activity (9,lO). Receptors coupled to stimulation of adenylyl cyclase have not been shown to work by this method E colz transformants should be picked and grown individually

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References 1. Yaghmai, R. and Hazelbauer, G. L. (1992) Ligand occupancy mimicked by single residue substitutions in a receptor: transmembrane signaling induced by mutation. Proc. Nat/. Acad, Sci. USA 89,7890-7894. 2. Ohphant, A. R. and Struhl, K. (1989) An efficient method for generating proteins with altered enzymatic properties: application to P-lactamase. Proc. Nat/. Acad SCL USA 86,9094-9098. 3. Arkin, A. P. and Youvan, D. C. (1992) Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis Bzorechnologv 10, 297-300. 4. Burstein, E. S , Spalding, T A , Hill-Eubanks, D , and Brann, M. R. (1995) Structurefunction of muscarimc receptor coupling to G proteins. J Biol. Chem 270,3141-3146. 5. Hill-Eubanks, D., Burstem, E. S., Spalding, T. A., Brauner-Osborne, H., and Brann, M. R. (1996) Structure of a G-protein-coupling domain of a muscarinic receptor predicted by random saturation mutagenesis. J. Biol Chem. 271,3058-3065. 6. Spalding, T. A., Burstein, E. S., Briiuner-Osborne, H., Hill-Eubanks, D , and Brann, M. R. (1995) Pharmacology of a constitutively active muscarmic receptor generated by random mutagenesis. J. Pharmacol. Exp Ther. 275,1274--1279. 7. Burstein, E. S., Spalding, T. A., and Brann, M. R. (1996) Amino acid side chams that define muscarinic receptor/G-protein couplmg. Studies of the third intracellular loop. J. Blol. Chem. 271,2882-2885. 8. Gutkind, J. S., Novotny, E. A., Brann, M. R., and Robbins, K. C. (1991) Muscarinic acetylcholine receptor subtypes as agonist-dependent oncogenes. Proc Natl Acad. Sci. USA S&4703-4707. 9. Brauner-Osborne, H. and Brann, M. R. (1996) Pharmacology of muscarinic acetylcholine receptor subtypes (ml-m5): high throughput assays in mammalian cells. Eur. J. Pharmacol 295,93-102. 10. Messier, T. L., Dorman, C. M., BrZiuner-Osborne, H., Eubanks, D., and Brann, M. R. (1995) High throughput assays of cloned adrenergic, muscarinic, neurokimn, and neurotrophin receptors in living mammalian cells. Pharmacol Toxicol. 76,30&3 11. 11. Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, Y.-C., and Axel, R (1977) Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell 11,223-232. 12. Lim, K. and Chae, C.-B. (1989) A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for P-galactosidase. BioTechniques 7,576-579. 13. Hermes, J. D., Blacklow, S. C., and Knowles, J. R. (1990) Searching sequence space by definably random mutagenesis: improving the catalytic potency of an enzyme. Proc. Natl. Acad Scl USA 87,696--700. 14. Murray, R., Pederson, K., Prosser, H., Muller, D., Hutchison, C. A., 3d, and Frelinger, J. A. (1988) Random oligonucleotide mutagenesis: application to a large protein coding sequence of a major histocompatibility complex class I gene, H-2DP. Nucl. Acids Res. 16,9761-9763.

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15. Derbyshire, K. M., Salvo, J. J., and Grindley, N. D. (1986) A simple and efficient procedure for saturation mutagenesis using mixed oligodeoxynucleotides. Gene 46,145-152. 16. Sambrook, J. (1989) A4oleculur Clonzng, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 17. Burstein, E. S., Spalding, T. A., and Brann, M. R. (1996) Constitutive activation of chimeric m2/m5 muscarmlc receptors and delineation of G-protein coupling selectivity domains. Biochem Pharmacol. 51,539-544. 18. Burstem, E. S., Spaldmg, T. A., Briiuner-Osborne, H., and Brann, M. R. (1995) Constitutive activation of muscarinic receptors by the G-protein G,. FEBS Lett. 363,26 l-263.

12 Identification and Quantitation of G Protein a-Subunits Ian Mullaney and Graeme Milligan 1. Introduction The demonstration that many intracellular signaling processes are mediated by a family of closely related guanine nucleotide binding proteins (G proteins) has led to the development of specific techniques that can be used to identify which of these polypeptide(s) is involved upon receptor activation by ligand. In addition, these methods can be used to probe the specificity of the interaction and to yield information about the stoichiometries involved. The classical G protems exist as heterotrimers composed of nonidentical a-, p-, and y-subunits. Initial identification of G proteins involved the ability of the a-subunit to act as a substrate for mono ADP-ribosylation catalyzed by the ADP-ribosyl transferase activity of a number of bacterial exotoxins, a modification that functionally altered the involvement of the G protein in signal transduction. The use of [32P]NAD as a substrate allowed the visualization of ADP-ribosylated polypeptides following separation in SDS-PAGE gels and autoradiography. In this way, G,, the G protein involved in the hormonal stimulation of adenylyl cyclase, was identified as a substrate for ADP-rtbosylation by cholera toxin. Similarly, G,, the inhibitory G protein, was characterized by its ability to act as a substrate for pertussis toxin. Although the use of toxins yields little information about the molecular identity of the G proteins involved, it can be useful in an initial investigation into G protein function m a particular system. However, not all G protein a-subunits are toxin substrates. The ability of agonists to activate phospholipase Cp and cause hydrolysis of inositol containing phospholipids has been shown to be unaffected by treatment with both pertussis and cholera toxins in the vast majority of cells and tissues studied, implying the involvement of toxin-insensitrve G proteins. From

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760 Table 1 Peptide Sequences Widely Used to Generate a Series of Antipeptide Antisera Directed Against the a-subunits of Various G Proteins

Peptideused RMHLRQYELLa KENLKDCGLFa LERIAQSDYI KNNLKECGLYa ANNLRGCGLYa GCTLSAEERAALERSK NLKEDGISAAKDVK

G protein sequence G,a 372-38 1 TD,a 341-350 G,2a 160-169 G,3a 345-354 G,a 345-354 G,a 1-16 G,a 22-35

QLNLKEYNLW

G,a 350-359

QENLKDIMLQ HDNLKQLMLQa QLNLREFNLV” ARYLDEINLL” QNNLKYIGLCa

G,,a 370-379 G,3a 368-377

Anttserurntdenttfies

G,,a 346-355 G,gx 365-374 G,a 346-355

aC-termmalsequences Amino acidsarerepresented usingthe oneletter code. TD, transducin.

Indeed, the use of polymerase cham reaction based on conserved sequence domains across the G protein family and the isolation of cDNAs has now identified more than 20 G protein a-subunits, including members of two new subfamilies (Gs and Glz) that are not substrates for the toxins. As a consequence of these studies, the primary amino acid sequences of all of these G protein a-subunits have been deduced, allowing for the generation of antipeptide antisera for use as specific immunological tools. Although the G protein superfamily is highly conserved, there are regions of sequence variatron, particularly m the carboxy terminal region that contains the receptor coupling sites, which have been successfully used to produce specific polyclonal antipeptide antisera (see Table 1). Such antisera are generally produced by subcutaneous injection of peptide conjugated to carrier protein into rabbits and can be tested either in ELISA assaysagainst the antigen peptide or by immunoblotting, using either purified or recombinant G protein a-subunits. The majority of G protein assays utilize cell-free systems, relying on the production of plasma membrane containing fractions from either tissue or cultured cells. Immunoblotting of supernatant fractions produced during membrane production generally shows little tmmunoreactivity, reinforcing the concept that the G protein a-subunits are located at membranes. All of the

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electrophoretic and immunoblotting techniques described herein use a crude plasma membrane preparation as starting material. All of the G protein a-subunits have predicted molecular masses of between 39 and 45 kDa. The gel conditions and sample treatments described have been optimized to separate polypeptides within this molecular mass range. Treatment of the samples with N-ethylmaleimide dtfferenttally alkylates the a-subunits of the pertussis toxin-sensitive G proteins G,1,G12,G,3,and the isoforms of G, on accessible cysteine residues. This has the effect of altering the migration of these a-subunits in SDS-PAGE with the result that it is possible to obtain greater resolution of the G, isoforms from the G,-like G proteins. If sample alkylation is performed in conjunction with resolution on a 12.5% acrylamide, 0.06% bisacrylamide gel, the separation achieved can be dramatic. This technique is particularly useful when trying to identify G proteins with antisera that crossreact, a common example being antisera directed against the carboxy terminus of G, can crossreact with G,3, because of the presence of an immunodominant tyrosine in the peptide sequence of these two polypeptides. To separate the isoforrns of G,CXor G proteins of the G, family, the best strategy we know of is to resolve the membranes on 12.5% acrylamide gels containing 6A4 urea. It is also possible to separate these proteins on SDS-PAGE gels, which contain a 4-&V urea gradient or on two-dimensional gels. However, both of these methods are technically more difficult and have little advantage over the 6M urea SDS-PAGE gel system. Sustained exposure of cell surface receptors to agonist frequently results in downregulation of both the receptor and the activated G protein. Pulse-chase assay techniques, in which ceils are first incubated with [35S]-methionine labeled medium then chased in the presence or absence of a receptor agonist and the G proteins subsequently immunoprecipitated with specific antisera, have revealed that the mechanism of downregulation is agonist-induced accelerated turnover of the a-subunit. The aim of this chapter is to describe the techniques, outlined above, that can be used to Identify and quantitate those G proteins involved in particular signaling events. 2. Materials 2. I. Equipment 1. 2. 3. 4. 5. 6.

Tissue culture facilities and shaking incubator. Sonicator. Benchtop centrifuge, microcentrifuge, and refrigerated ultracentrifuge. ELISA plate reader. Tight fitting teflon-on-glass homogenizer or polytron. 100°C heating block.

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SDS-PAGE gel apparatus and power pack. Electroblotting apparatus and power pack. Rotating wheel. Gamma counter or phosphorimager. Imaging densitometer. Gel drier and vacuum pump. X-ray film developing apparatus

2.2. Reagents 1. Keyhole limpet hemocyanin, Freund’s complete and incomplete adjuvants. 2. Glutaraldehyde: 2 1 mM glutaraldehyde in H20. 3. Phosphate-buffered saline (PBS): 0.2 g KCl, 0.2 g KH2P04, 8 g NaCl, 1.14 g Na,HPO, (anhydrous), pH 7.4, up to 1000 mL with HzO. If kept as a stock, check pH before use 4 Peptrde solution (10 pg/mL). Dilute 1: 100 from 1 mg of peptide in 1 mL of PBS Stock can be stored at -20°C. 5. ELISA blocking solutron: 1 g dried milk in 100 mL PBS. 6. PBS-Tween-20: 0.5 mL Tween-20 in 1000 mL PBS. 7. ELISA antibody carrier solution: 0.05 mL Tween-20,0.1 g dried rmlk in 100 mL PBS. 8. ELISA primary antibody solutton. G protein antisera diluted m ELISA antibody carrier solutron. 9. ELISA secondary antibody solution: 1: 1000 dilution of horseradish peroxidaseconjugated donkey antirabbit IgG diluted m ELISA antibody carrier solution. 10 Cttrate phosphate buffer: buffer A: 0. 1M citric acid, 2 1.O1 g m 1000 mL H20; buffer B: 0.2MNa2HP04 (anhydrous), 28.4 g in 1000 mL H20. Mix 17.9 mL buffer A, 32.1 mL buffer B, 50 mL HZ0 adjust to pH 6.0. Buffers A and B can be stored at 4°C. 11. HzOz solution: 10 pL of HzO, m 10 mL HZ0 (prepare fresh as needed) 12. OPD substrate solution: 4 mg OPD (o-phenylenediamide dthydrochloride), 9 mL citrate phosphate buffer, 1 mL H,O, solution. 13 L-broth 10 g tryptone, 5 g yeast extract, 10 g NaCl to 1000 mL with HZ0 (pH 7.0 with NaOH), sterilize in a pressure cooker (e.g., Prestige medical series 2100 clinical autoclave). 14. L-broth/glucose: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 3.6 g glucose to 1000 mL with HP0 (pH 7.0 with NaOH); sterilize in a pressure cooker. 15. Ampicillin (50 mg/mL stock): 0.5 g in 10 mL H,O; filter sterilize and store as 1-mL aliquots at -20°C. 16. L-broth-agar: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar to 1000 mL with Hz0 (pH 7.0 with NaOH); sterilize in a pressure cooker. When hand cool, add 2 mL ampicillin stock, shake to mix, and pour plates (approx 10 mL per plate). 17. IPTG (100 mM): 0.476 g up to 20 mL H20, filter sterilize and store as 2-mL aliquots at -20°C. 18. TE buffer (10 mMTris-HCl, 0.1 mMEDTA, pH 7.5): 1.21 g Tris, 37.2 mg EDTA in 1000 mL H20; adjust to pH 7.5.

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19. Protease inhibitors: TE buffer contaming 10 mil4 NaF, 100 @4 NasVO,, 1 mM phenylmethanesulfonyl fluoride, 3 mM benzamidine, 0.1 piW soybean trypsin inhibitor, 10 p/t4 leupeptm, 0.2 @4 aprotinin, 1.5 @4 antipain. 20. 2% (w/v) 7-deoxycholw acid, sodium salt: 2 g in 100 mL H,O. 21. 24% (w/v) TCA: 24 g in 100 mL H,O. 22. IM Tris base: 121.1 g in 1000 mL HzO. Do not adjust the pH. 23. Laemmli sample buffer: 0.605 g Tris, 30 g urea, 5 g SDS, 6 g DTT, 10 mg bromophenol blue in 100 mL H20, pH adjusted to 8.0 with HCl. Aliquot in 2 mL vol and store at -20°C 24. 5% (w/v) SDS, 50 mM DTT: 5 g SDS, 0.771 g DTT in 100 mL H,O. 24. 100 WNEM: 1.25 g m 100 mL H,O. 25. Cholera toxin (Sigma, Poole, UK, product no. C-3012): kept as a 1 mg/mL stock at 4°C in 50 mM Tris-HCl (pH 7.5), 200 mMNaC1,3 mMNaN,, 1 mM EDTA. 26. Pertussis toxin (Porton, Porton Down, UK): stored at -2O’C as a 0.44 mg/mL stock in 50 Wphosphate buffer (pH 7.2), 500 mA4NaC1,50% (v/v) glycerol. 27. Nicotinamide adenine dinucleotide, di(triethylammonium) salt, [adenylate-32P] (product no NEG-023): sp acttvity 10-50 Ci/mmol from NEN Research Products, Du Pont (UK), Stevenage, UK. 28. 1.5M sodium phosphate buffer: 53.25 g Na2HP0, in 250 mL H20 (solution 1); 58.5 g NaH,PO, * 2H,O in 250 mL H20 (solution 2). Titrate solution 2 mto solution 1 until pH 7.0 is reached. 29. ADP-ribosylation cocktail stocks: 0.2Mthymidine, 48.44 mg/mL H20; lMarginine HCI, 210.7 mg/mL HzO; 0.04M ATP, 22.04 mg/mL H20, 1 mM GTP, 0.54 mg/mL H20. Store in 100~@., aliquots at -20°C 30. ADP-ribosylation cocktail mix (for 15 samples): 75 pL thymidine, 30 pL GTP, 125 pL 1.5M sodturn phosphate buffer, 10 pL ATP, 15 pL argmine hydrochloride, 30 pCi [32P] NAD+ to 300 pL H20. 3 1. 35Slabeling medium: methionine and cysteine free Dulbecco’s modified Eagle’s medium (ICN Bromedicals [Thame, UK]) supplemented with 50 pCi/mL Tran3?S-label (ICN) and 1% (v/v), heat inactivated, dialyzed fetal bovine serum. 32. 10% (w/v) SDS: 100 g SDS in 1000 mL H20. Keep at room temperature and use as the stock for all SDS solutions. 33. Solutions for 10% (w/v) SDS-PAGE gels: resolving gel: a. Acrylamide: 30 g acrylamide, 0.8 g brsacrylamide to 100 mL wtth H20 (see Note 1); b. Gelbuffer 1: 18.17gTris,4mL lO%(w/v)SDS(pH8,8)to 100mLwithH20; c. Glycerol: 50% (v/v) in H,O; d. APS: 100 mg ammonium persulfate per mL HzO. 34. Solutions for 10% (w/v) SDS-PAGE gels: stacking gel: Gel buffer 2: 6 g Tris, 4 mL 10% (w/v) SDS (pH 6.8) to 100 mL with H20. 35. Solutions for 12.5% (w/v) SDS-PAGE gels: resolving gel: Acrylamide: 30 g acrylamide, 0.15 g bisacrylamide to 100 mL with H20. 36. Electrophoresis running buffer: 6 g Tris, 28.8 g glycine, 20 mL 10% (w/v) SDS to 2000 mL with H20. Do not adjust pH.

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37. 6Murea: 36.03 g urea up to 100 mL with Hz0 Make fresh on day of use. 38. Solutions for 12.5% (w/v) SDS-urea PAGE gels: resolving gel: a Acrylamide: 30 g acrylamide, 0.15 g bisacrylamide, 36.025 g urea to 100 mL with H,O; b. Gel buffer 1: 18.17 g Tris, 36.025 g urea, 4 mL 10% (w/v) SDS (pH 8.8) to 100 mL with H,O; c. Glycerol: 50% (v/v) in H20; d APS: 100 mg ammomum persulfate per mL Hz0 39. Coomassie stain buffer: Dissolve 2.5 g Coomassle brilliant blue R-250 into a solution containing 450 mL methanol, 450 mL H,O, 100 mL glacial acetic acid Filter through Whatman No. 1 filter paper. This solution can be reused. 40 Gel destain buffer: 450 mL methanol, 450 mL H,O, 100 mL glacial acetic acid. 41. Blotting buffer: 15 g Tris, 72 g glycine, 1000 mL methanol to 5000 mL with H,O. Do not adjust pH. 42 Ponceau S solution: 15 g tnchloroacetic acid in 500 mL H,O, allow to dissolve and add 0.5 g Ponceau S. Keep stock solution at room temperature and reuse. 43 Immunoblot blocking buffer: 5 g gelatin in 100 mL PBS. 44. PBS-NP40: 2 mL Nonidet P40 in 1000 mL PBS. 45 Immunoblot first antiserum solution: appropriate anti-G protein antlserum dilution m PBS-NP40 containing 1% (w/v) gelatin. 46. Immunoblot second antibody solution. 1:500 dilution of a commercial horseradish peroxidase-conjugated donkey antirabbit IgG in PBS-NP40 containing 1% (w/v) gelatin. 47. o-diansldine solution: 10 mg o-diansldine hydrochloride solution m 1 mL Hz0 (see Note 2). 48. Sodium azide solution: 1 g NaN3 m 100 mL Hz0 (see Note 3). 49. IP buffer: 6.06 g Tris, 11.1 g NaCl, 2 23 g EDTA, 12.5 mL Triton X- 100 (pH 7.5) to 1000 mL with H20. 50. IP wash buffer: 80 mL IP buffer, 20 mL 1% (w/v) SDS. 51 IP final wash buffer: 0.606 g Tris (PH 6.8) to 100 mL HzO. 52. [ 1251]-labeled donkey antirabbit immunoglobulm (product no. IM 134)-sp. actlvity 750-3000 Cl/mm01 (Amersham International, Amersham, UK). 53. [‘251]-overlay solution* 50 mL PBS-NP40 containing 1% (w/v) gelatm and spiked with 5 pCi [1251]-labeled donkey anttrabbit immunoglobulin.

3. Methods 3.7. Production of G Protein-Specific Antisera 3. I. 1. Immunization and Serum Collection Antipeptide antisera are prepared according to the method described by Goldsmith

et al. (11, in commercially

purchased New Zealand White rabbits.

Preimmune blood samples should be taken from each of the animals prior to injection and the serum should be checked for any significant titer or immunological ldentlficatlon of cellular proteins.

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1. Dissolve 10 mg of keyhole limpet hemocyanin and 3 mg of the particular peptide (see Note 4) required in 1 mL of 0. 1MNa phosphate buffer (pH 7.0). Add 0.5 mL of 21 mMglutaraldehyde dropwise with stirring and incubate the combined 1.5 mL overnight at room temperature. 2. Mix with an equal volume of Freund’s complete adjuvant and sonicate for 20 s. 3. Immediately after sonication, inject the resultant emulsion in 0.2~mL aliquots into multiple subcutaneous sites in the rabbit Immunizations are normally performed simultaneously mto two rabbits in order to maximize successful antibody production. 4. After 2 wk, each animal receives a booster immuruzation with material prepared identically, except that one-half as much peptide and KLH are injected in Freund’s incomplete adjuvant. 5. Four weeks after the booster injections, bleed the animals. Collect the blood into glass universals and allow to clot overnight at 4°C (see Note 5). 6. Remove the straw-colored serum from the clot and centrifuge at 1000 rpm for 5 min on a benchtop centrifuge to remove any remaining traces of erythrocyte Aliquot the serum m appropriate volumes and store at -20°C 3.12.

ELISA

Protocol

1. Coat a 96-well titertek ELISA plate with antigen peptide. Add 100 & of 10 pg/mL peptide solution, cover plate with cling film, and incubate overnight at 4’C (see Note 6). 2. Remove liquid and wash each well two times with PBS (see Note 7). 3. Blot dry, add 100 @, of ELISA blocking solution to each well, and incubate at 37’C for 1 h. 4. Remove blocker, wash each well twice with PBS-Tween-20, and blot dry. 5. Add 100 pL of increasing dilutions of antiserum (from 1: 10 to 1: 100,000) to each well, cover plate with cling film, and incubate overnight at 4°C (see Note 8). 6. Remove antiserum dilutions, wash each well twice with PBS-Tween-20, and blot dry. 7. Add 100 pL of secondary antibody solution to each well and incubate at 37OCfor 1 h. 8. Remove secondary antibody solution, wash each well rive times with PBSTween-20, and blot dry. 9. Add 100 mL of OPD substrate solution to each well, wrap the plate in tin foil, and incubate, in the dark, at room temperature for 15-20 min (see Note 9). 10. Stop reaction by addition of 50 pL 2MH,SOQ to each well and read at 492 nm on any ELISA plate reader, e.g., Titertek Multtscan. 11. Plot the absorbency reading against antiserum dilution to determine specificity (see Note 10).

3.2. High-Level Expression of Mammalian G Protein a-Subunits in Escherichia coli The method described below uses plasmid pT7.7, into which the various Ga genes have been subcloned (Wise and Milligan [2/). This expression system

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utilizes the promoter for bacteriophage T7 RNA polymerase m the plasmid vector pT7.7. This expression vector contains an initiation codon ATG and a ribosome binding site positioned downstream from the T7 promoter so that maximal expression is ensured. Expression constructs were transformed into the lysogen BL21 (DE3), which contains a single chromosomal copy of the gene for T7 RNA polymerase under control of the isopropyl+-p-thiogalactopyranoside (IPTG)-inducible luc UV5 promoter. 3.2.1. Preparation of Competent E. coli Strain BL27 DE3, Transformation of Cells with the Expression Vector pT7.7 and Expression of Mammalian G Protein a-Subunits 1. Take 50 pL of E. colz stock, add 5 ng of plasmid DNA, and Incubate on ice for 15 min

2. Heat the cells at 42°C for exactly 90 s, then return to ice for 2 mm.

3 Add 450 pL of L-broth/glucoseand allow cells to recover by incubation at 37OC for 1 h in a shakmgincubator. 4. Spread100pL of the transformantsonto L-broth-agarplatescontaming 100pg/mL ampictllin and incubate overnight at 37°C. 5 Select single colonies from the plate and put into 10 mL L-broth containing 100 pg/mL amprcillrn and incubate in a shaking incubator overnight at 37°C. 6. Take 0 5 mL of the overnight culture, inoculate mto 50 mL L-broth containing 100 pg/mL. ampicillin, and incubate in a shaking incubator at 37°C until an A550 of between 0.3 and 0.5 absorbency units is reached. 7. Remove 1 mL of the cell suspensron as a control, add 0.5 mL of 100 mMIPTG to the remainder, and incubate m a shaking incubator at 37°C for 4 h. 8. Remove 1 mL of cell suspension, spin m a benchtop mtcrocentrifuge for 10 mm at 12,000 rpm, and discard the supernatant

9. Add 25 pL of Laenunli samplebuffer to the pellet, heat to 90°C for 10min, and resolve the whole-cell extracts by SDS-PAGE (see Fig. 1).

3.3. Sample Preparation for Analysis of G Proteins 3.3.7. Production of Crude Plasma Membrane Fractions Crude plasma membrane fractions are produced essentially as described by Koski and Klee (3). 1. Gently remove cells off the surface of the flasks with a Pasteur pipet or rubber

policeman, collect in a 50-mL conical centrifuge tube on ice, and centrifuge at 1000 rpm at 4°C for 5 min on a benchtop centrifuge. 2. Discard the supernatant, resuspend the cell pellet in 30 mL me-cold PBS, and

centrifuge asbefore. 3. Repeat this procedure twice and store the resultant washed cell paste at -8O’C

until needed.

167

G Protein a-Subunits Mr

12 Fig. 1. Expression of recombinant G,a in E. coli BL21 (DE3) cells. Cultures of transformed cells were induced to express recombinant Ggo, harvested, and resolved on a 12.5% acrylamide, 0.06% bisacrylamide gel, as described in Sections 3.2.1. and 3.4.2. 10 pL of whole-cell extract from uninduced cells (lane 1) and induced cells (lane 2) were resolved and visualized by staining with Coomassie blue.

4. Thaw the frozen cell pastes and resuspend in 2 mL of ice-cold TE buffer and homogenize with 20 strokes of a teflon-on-glass tissue grinder (see Note 11). 5. If preparing plasma membrane fractions from tissues, chop the tissue with scissors, rinse in two washes of PBS (see Note 12), and homogenize for 60 s in 10 vol of TE buffer with a polytron at setting number 4. 6. Centrifuge the homogenates for 10 min at 500g in an ultracentrifuge (a Beckman L5-50B for example). 7. Discard the pellet (see Note 13) and centrifuge the supernatant, which will contain the bulk of plasma membrane fraction, at 48,000g for 10 min, using the same centrifuge. 8. Discard the supernatant, resuspend the pellet in 5 mL of TE buffer, and recentrifuge for 10 min at 48,OOOg. 9. Again, discard the supernatant and resuspend the pellet in TE buffer. 10. Triturate the resuspended pellet with a syringe with a tine gage needle. Aliquot into appropriate volumes and store at -8O’C until needed. 11. Protein concentrations are determined. Membranes should have protein concentrations of l-2 mg/mL.

168 3.3.2. TCA/Deoxycho/ate

Mullaney and Milligan Precipitation of Samples

1. Take an approprtate amount of the crude membrane preparation (between 25 and 150 pg of membranes, depending on the experiment) and place on ice m a 1S-mL Eppendorf centrifuge tube. Samples with a volume of over 100 pL should be spun for 5 mm at 12,000 r-pm on a microcentrifuge, the supematant carefully removed, and the pellet resuspended m 20 pL of TE buffer. 2. Add 6.5 pL of 2% (w/v) 7-deoxychohc acid, sodtum salt to each tube, followed by 750 pL of double-distilled H20, then 250 pL 24% (w/v) TCA (see Note 14) 3. Vortex mix each sample and spm in a benchtop microcentrifuge for 10 mm at 12,000 rpm 4 Carefully discard the supematant (see Note 15) and bring the pellet to weakly alkaline pH by addition of 20 pL 1M Tris base. 5 Add 20 pL Laemmli sample buffer (see Note 16) and load sample onto gel

3.3.3. NEM Treatment Alkylation of samples prior to electrophoresis may produce better resolution of G proteins with very similar molecular masses. 1. Take an appropriate amount of the crude membrane preparation (between 25 and 150 pg of membranes, depending on the experiment) and place on ice in a 1.5-r& Eppendorf centrifuge tube. 2. Centrifuge the samples at 12,000 rpm for 5 mm on a benchtop microcentrifuge. 3. Remove the supematant and resuspend the pellet in 20 Ccs,TE buffer. 4. Add 10 pL of 5% (w/v) SDS, 50 mA4 DTT and incubate at 90°C for 5 min 5 Cool the samples on ice, add 10 pL of freshly prepared 100 mM NEM to each tube, and leave at room temperature for 20 min. 6. Add 20 pL Laemmli sample buffer and load sample onto gel.

3.3.4. Mono-ADP Ribosylation of Membranes by Bacterial Toxins Mono-ADP ribosylation of G proteins for further analysis by SDS-PAGE is derived from the method of Hudson and Johnson (4). 1. Both cholera and pertussis toxins must be preactivated before in vitro use. Add an equal volume of 100 mM DTT to the toxin, gently mix, and sit at room temperature for up to 1 h (see Note 17). 2. Take an appropriate amount of the crude membrane preparation (between 25 and 50 pg of membranes m a final volume of 25 pL) and place on ice m a 1.5~mL Eppendorf centrifuge tube. 3. Add 20 pL of the rtbosylation cocktail mix to each tube and start the mcubation by adding 5 pL of the appropriate preacttvated toxin (see Note 18) Incubate for up to 90 mm in a 37°C water bath. 4. Place on ice and precipitate samples using the TCA-deoxycholate method (see Section 3.3.2.). 5. Add 20 pL Laemmli sample buffer and load sample onto gel (see Note 19).

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169

3.3.5. p5S]-Trans Pulse-Chase Protocol 1. Seed cells into 75-cm3 flasks or 6-well culture dishes 2. When cells are approx 60% confluent, replace growth medium with 35Slabeling medium (see Note 20). 3. Incubate cells in 35S-label for 20-48 h (pulse). 4. Wash cell monolayer twice with normal growth medium and leave in fresh growth medium (see Note 2 1) (chase). 5. At appropriate ttmes, gently wash cells off the surface of the flask with a Pasteur pipet or rubber policeman, collect m a 12.5-mL plasttc centrtfuge tube on ice, and centrifuge at 1000 rpm at 4’C for 5 min on a benchtop centrifuge. 6. Take off medium, resuspend the cells in 100 pL H20, and add 100 pL 2% (w/v) SDS (see Note 22). Transfer to a 2-mL screw-cap Eppendorf tube. 7. Tighten screw-cap onto tube and heat to 100°C for 20 min (see Note 23) 8. Transfer tube to ice, pulse spin to collect all moisture to bottom of tube, and proceed to the immunoprecipitation protocol (see Note 24).

3.4. SDS-Polyacrylamide Gels are performed

Gel Electrophoresis

(SDS-PAGE)

using the basic approach of Laemmh

(5) on slab gels.

3.4.1. 10% Acrylamide (w/v) SDS-PAGE Gels Gel plates (180 x 160 mm, with spacers of 1.5 mm) are run as a part of a Bio-Rad Protean I electrophoresis apparatus (Bio-Rad, Watford, Herts., UK). 1. Recipe for one gel: resolving gel (lower) solutions: 8.2 mL H20, 6 mL Buffer 1, 8 mL Acrylamrde, 1.6 mL 50% Glycerol, 90 pL APS, 8 pL TEMED. 2. Set up the gel apparatus according to the manufacturer’s guidelines Add all reagents in the order given into a 250 mL conical flask and mix gently Cast gel using Pasteur pipet (see Note 25). 3. Carefully overlay cast gel with approx 1 mL of 0.1% (w/v) SDS and allow gel to polymerize (between 1 and 2 h at room temperature). 4. After polymerization, remove SDS overlay, and prepare to add stacker gel. Stacker gel (upper) solutions: 9.75 mL H20, 3.75 mL Buffer 2, 1 5 mL Acrylamide, 150 pL APS, 8 pL TEMED 5. Add all reagents in the order given into a 100~mL conical flask and mix gently. Pour stacker gel on top of resolving gel and place well forming comb m top of gel, ensuring no air bubbles are trapped under the comb. Leave to polymerize (approx 1 h at room temperature). 6. After polymerization, remove sample well comb and place the gel m the gel tank containing enough running buffer in the base to cover the bottom edge of the gel and add the remaining runnmg buffer to the top. 7. Load the prepared samples m the preformed wells, using a Hamilton syringe. 8. Run the gel overnight (approx 16 h) at 60 V and 15 mA per plate until the dye front reaches the bottom of the gel plates.

170

Mullaney and Milligan

3.4.2. 12.5% Acrylamide (w/v) SDS-PAGE Gels (Gel plates 180 mm x 200 mm, with spacers of 1.5 mm.) When higher resolution of proteins within a narrow mol wt range is needed, 12.5% (w/v) SDS-PAGE gels should be run, instead of 10% (w/v) gels. The longer gels are able to maxtmize detection of molecular mass differences and are run as part of a Bra-Rad Protean II electrophoresis system 1. 1. Recipe for one gel: resolving gel (lower) solutions: 11.6 mL H20, 12 mL Buffer 1,20 mL Acrylamtde, 4 mL 50% Glycerol, 180 pL APS, 16 pL TEMED. 2 Set up the gel apparatus according to the manufacturer’s guidelines Add all reagents in the order given into a 250~mL conical flask and mtx gently. Cast gel using Pasteur pipet (see Note 25) 3 Carefully overlay cast gel with approx 1 mL of 0.1% (w/v) SDS and allow gel to polymerize (between 1 and 2 h at room temperature). 4. After polymerization, remove SDS overlay and prepare to add stacker gel. Stacker gel (upper) solutions: 9 75 mL H,O, 3 75 mL Buffer 2, 1.5 mL Acrylamtde, 150 pL APS, 8 pL TEMED. 5. Add all reagents m the order given mto a lOO-mL comcal flask and mix gently Pour stacker gel on top of resolving gel and place well forming comb in top of gel, ensuring no an bubbles are trapped under the comb. Leave to polymertze (approx 1 h at room temperature). 6 After polymerization, remove sample well comb and place the gel m the gel tank containing enough runnmg buffer m the base to cover the bottom edge of the gel and add the remaining runnmg buffer to the top 7 Load the prepared samples in the preformed wells, using a Hamilton syringe 8. Run the gel overnight (approx 16 h) at 100 V and 15 mA/plate unttl the dye front reaches the bottom of the gel plates

3.4.3. 12.5% Acrylamide (w/v) SDS-Urea-PAGE

Gels

Resolutton of many closely related G protein a-subunits can be achieved usmg 12.5% SDS-PAGE gels in which 6M urea has been added to the gel mixture (see Note 26). Gel plates (180 x 160 mm, with spacers of 1.5 mm) were run as a part of a Bio-Rad Protean II electrophoresis apparatus (see Fig. 2). 1. Recipe for one gel: resolvrng gel (lower) solutrons 11.6 mL 6M urea, 12 mL 6M urea/Buffer 1, 20 mL 6M urea/Acrylamide, 4 mL 50% Glycerol, 180 pL APS, 16 p.L TEMED. 2. Set up the gel apparatus according to the manufacturer’s guidelines Add all reagents m the order given into a 250-n& conical flask and mix thoroughly. Cast gel using Pasteur prpet (see Note 25). 3. Carefully overlay cast gel wrth approx 1 mL of 0.1% (w/v) SDS and allow gel to polymerize (3-4 h at room temperature) (see Note 27) 4. After polymerization, remove SDS overlay and prepare to add stacker gel. Since there is no urea in the stacker gel, follow the procedure given for nonurea-contaming gels.

171

G Protein a-Subunits

-Gil Gil (1 d

+Gi2tx

(x -ADP ribose -ADP ribose

G,2
1

2

Fig. 2. Separation of G protein a-subunits on SDS-urea PAGE gels. Membranes (50 pg) prepared from untreated cos 7 cells (lane 1) and cos 7 cells pretreated with pertussis toxin (100 ng/mL growth medium, 16 h) (lane 2) were resolved on SDS-urea gels and immunoblotted with an antiserum that recognizes the a-subunits of Gi 1 and Gi2 as described in Sections 3.4.3., 3.5.1., and 3.5.2. 5. After polymerization, remove sample well comb and place the gel in the gel tank containing enough running buffer in the base to cover the bottom edge of the gel, and add the remaining running buffer to the top. 6. Load the prepared samples in the preformed wells, using a Hamilton syringe, 7. Run the gel for 18-20 h at 120 V and 50 mA/plate (see Note 28).

3.4.4. Autoradiography 1. Remove SDS-PAGE gel with resolved radiolabeled polypeptides from the electrophoresis apparatus and soak for 1 h in Coomassie stain buffer. 2. Remove stain, add destain solution, and leave for 2-3 h. 3. Remove stain and dry the gel onto Whatman 3MM filter paper under suction from an electric vacuwn pump attached to a gel drier (e.g., a Bio-Rad model 583) at 70°C for 2 h. 4. Transfer the dried gel to a Kodak X-o-matic cassette with intensifying screens (or similar) containing Kodak X-omat S X-ray film and allow to autoradiograph at -80°C for an appropriate time. 5. Develop the film (e.g., on a Kodak X-o-mat developing machine) and quantitate the autoradiograph using an imaging densitometer.

3.4.5. Phosphorimaging l-3. Follow steps l-3 in Section 3.4.4. 4. Transfer the dried gel to a phosphorimager plate and leave for an appropriate length of time. 5. Develop the image using a phosphorimager (e.g., Fujix BASlOOO) and quantitate the resultant image.

Mullaney and Milligan

172 3.5. Immunological

Methods

Transfer of proteins from SDS-PAGE gels onto nitrocellulose and subsequent incubation with antisera essentially follows the strategies reported by Towbin (6). 3.5.1. Electroblotting of Proteins onto Nitrocellulose Proteins that have been separated on SDS-PAGE gels are transferred onto mtrocellulose using electroblotting apparatus (e.g., an LKB 2005 Transphor unit). 1, Soak the sponge pad in blotting buffer and place m the lower part of the transfer cassette.All subsequent loadmg procedures are done with the cassette

2. 3.

4. 5.

6.

totally submerged m blotting buffer to prevent formation of any air bubbles within the cassette, which prevents successful transfer of the proteins from the gel. Place a piece of Whatman 3MM chromatography filter paper (with dimensions shghtly larger than the gel) on the sponge and put the gel on top Position a piece of nitrocellulose, cut approximately to the size of the gel, over the gel, then another piece of Whatman 3MM filter paper, and finally another sponge to completethe sandwich. Close the cassette, insert into the transfer apparatus, and electroblot toward the anode at 1SA for 2 h (see Note 29). To check if transfer is complete, remove nitrocellulose from the blotting sandwich, place m a clean container, and cover with Ponceau S solution. Gently rock until protein banding appears. Discard staining solution and wash blot with blotting buffer from the electroblotting tank until the bands disappear.

3.5.2. Incubation of Nitrocellulose with Antisera 1. Transfer the electroblotted mtrocellulose sheetinto a dish, cover with 100 mL immunoblot blocking buffer, and incubate for 2 h at 3O’C. 2. Remove blocker, wash the nitrocellulose using copious amounts of double-dutilled water, add the first antiserum solution (normally the specific anti-G protein antiserum), and incubate overnight at 30°C (see Note 30). 3. Remove primary antiserum and wash the blots thoroughly with double-distilled water to remove all the unbound antiserum. 4. Wash the blot with PBS-NP40 for 2 x 10 min, then incubate at 30°C for 2 h in the second antiserum solution. 5. Thoroughly wash the blot with double-distilled water, then with PBS-NP40 for 2 x 10 min, and finally with two washes of 10 min each with PBS. 6. Place blot in dish containing 40 mL PBS. Add 1 mL newly prepared o-diansidme solution, then 10 & of stock hydrogen peroxide.

G Protein a-Subunits

173

7. Remove developer, terminate reaction by addition of sodium azlde solution (see Note 3 l), and leave for 2 min. Pour off sodium azide and wash with water.

3.5.3. lmmunoprecipitation

Protocol

1. To each sample in a final volume of 50 pL, add 150 pL of 1.33% (w/v) SDS, containing the cocktail of protease inhibitors previously described, in 2-mL screw-top Eppendorf tubes (see Note 32). 2. Heat the samples to 1OO“C for 4 mm, place on ice to cool, and pulse the samples to the bottom of the tubes by briefly spinning the samples at maximum speed in a microcentrifuge. 3. Add 0.8 mL of ice-cold IP buffer containing protease inhibitors to each sample and mix by inverting. 4. Spin samples in a microcentrifuge at 12,000 rpm for 10 min at 4“C and transfer supematant to a new tube. 5. Add an appropriate amount of antibody (between 2 and 20 pL, dependmg on the antiserum) and incubate with rotation at 4OC overnight. 6. Add 50 pL of Pansorbin (or 20 pL protein A Sepharose) (see Note 33) to each sample and incubate at 4OC, with rotation for a mimmum of 4 h 7. Spin samples in a mtcrocentnfuge at 12,000 rpm for 2 min at 4’C and wash with 3X 1 mL IP wash buffer, pelleting the Pansorbin complex between each wash by centrifugation for 30 s at 12,000 rpm. 8. After the final spin, remove the supematant and wash the pellet wtth 1 mL IP final wash buffer. 9. Repeat centrifugatlon step. 10. Remove final wash buffer and add 50 & Laemmh buffer Heat samples to 100°C for 10 mitt, centrifuge for 10 min at 12,000 to pellet Pansorbin, then load supernatant on gel.

3.6. Quantitation of G Protein a-Subunits This is achieved by immunoblotting various amounts (O-100 ng) of either E co&expressed G protein (see Section 3.2.) or purified G protein, along with known amounts of the plasma membrane fractions. A standard curve can be constructed in the following ways, and levels of G protein in the sample can be assessedand expressed in terms of membrane protein, tissue amount, or even cell number. Commercial preparations of partially purified G protein a subunits are available (see Note 34). 3.6.1. Densitometric Quantitation of lmmunoblots 1. Place the developed immunoblot onto filter paper and allow to dry. 2. Scan the blot into an imaging densitometer (e.g., Bio-Rad GS-670) and quantitate. 3. Plot the standard curve showing amount of recombinant or purified G protein against arbitrary densitometric values and extrapolate the values for the unknowns from the curve (see Ftg. 3).

Mullaney and Milligan

174

A

2.5

5

7.5

10

15

20

30

GsCt long (ng)

*O” B

0 0

I 10

1

20

I 30

Gsa long (na)

Fig. 3. Quantitation of levels of E. co&expressed Gsa. (A) O-25 ng of E. coliexpressed G,a (long form) were resolved by SDS-PAGE and immunoblotted with an antiserum directed against the a-subunit of G,, as described in Sections 3.4.1.) 3.5.1, and 3.5.2. (B) The immunoblot was subjected to densitometic analysis and a standard curve was constructed as described in Section 3.6.1.

3.6.2. ~251]-Labeled Donkey Antirabbit lmmunoglobulin Overlay Technique 1. Place the developed blots in a dish containing 50 mL [*251]-overlay solution and incubate for 1 h at 30°C.

G Protein a-Subunits

175

2. Remove the overlay solution, wash the blots thoroughly with double-drstilled water to remove all the unbound label, and then with two washes of 30 min each with PBS. 3. Allow the blot to air-dry, excise the immunoreactive bands, and measure by liquid scintillation counting. 4. Plot the standard curve showing amount of recombmant or purified G protein against the dpm obtained from the bound [1251]-overlay solution and extrapolate the values for the unknowns from the curve

4. Notes 1. Care should be taken with acrylamtde and bisacrylamrde, since both have been reported to be neurotoxms 2. Dissolve o-dransidine hydrochloride in H20, because rt is poorly soluble in PBS. 3. NaN, is toxrc. The solution can be reused but tends to turn brown and discolor the mtrocellulose 4. See Table 1 for peptrde sequences used m G protein antipeptide antisera production. 5. Do not use plastic universals. Glass universals allow the clot to shrink, enabling serum harvest. 6. It is advisable to test any antisera produced against other peptides, e.g., other C-terminal G protein sequences and nonrelated peptrdes to examme for nonspecific immunoreactivity. 7 For simplicity, fill water bottles with PBS or PBS-NP40 and use these to wash the ELISA plates, 8. Dilute the antiserum simply by a series of 1: 1 dilutions, starting from 1.10, 20, 40,80, 160, and so on 9. Positive reaction is purple, which turns to brown upon addrtron of H2S04. If no color appears after 1 h, mix the remaining secondary antibody solution with the remaining OPD solution. If the solutions are correct, a purple color will appear instantaneously. ’ 10. Typical half maximal antiserum dilutions for useful antisera are 1: lO,OOO1*100,000. 11. Protease inhibitors should be mcluded in all buffers. 12. The easiest way is to mince the tissue on a plastic Petri dish with curve-ended scissors before disruption 13. Depending on the tissue or cell line use, the pellet after the first spin can be large. It is advisable to keep this pellet on ice until the plasma membrane fractionation 1scomplete, in case the original homogenization was unsuccessful. 14. The solutions must be added in the order 7-deoxycholate, H20, then TCA. Solutions can be kept indefinitely at room temperature. 15. Remove the supematant and blot dry on to tissue to get rid of as much liquid as possible. The pellet should be firmly sedimented to the bottom of the tube. 16. If the blue Laemmli buffer solution turns brown/orange, then the sample is still acidic. Simply add 5-pL amounts of 1MTris base until the sample turns blue. 17. To treat cells in vivo with pertussis or cholera toxin, incubate in growth medium containing 100 ng/mL toxin for 16 h and harvest the cells as described.

Mullaney and Milligan 18. If a number of samples contain the ribosylation cocktail and the toxin, then mix the two solutions together in the ratio of 4: 1 and add 25 pL, to each sample. This will avoid sample variation caused by additton of small volumes. 19 As these samples are radioactive, load the gel using an automatic pipet and dispose of the tips along with the rest of the radioactive waste. 20. Caution: 35Sis volatile. Stocks should be opened and aliquoted in a fume hood Store aliquots m 2-mL screwtop Eppendorf tubes and store at -80°C. 2 1. This is the chase element in pulse-chase protocols. 22. To ensure proper solubtlization of the cell pellet, always resuspend in H,O, then add the SDS. Do not resuspend the cell pellet directly in SDS. 23. If the sample IS still viscous at this stage, pass it through a 25-gage needle and syringe and reboil as before for 10 mm. 24. At this stage, it is possible to freeze the sample at -20°C and store until further use 25 There is no need to degas the solutions before pouring the gel 26. Although we have reported that separation of certain G protein a-subunits (such as G,a from G, ia) can best be achieved on 4-8M urea gradient gels, our more recent experience suggests that there is httle advantage over the 6M urea gels, whtch are considerably caster to set up. 27. If the room temperature is on the cold side, there is the possibility that the urea will come out of solution as the gel is polymerizing. To avoid this, allow the gel to set in warmer rooms, e.g., tissue culture room, 30°C hot room, and so on. 28. Polypeptides do not run normally on SDS-urea gels. They may appear to run at different molecular masses than in the absence of urea, making it more difficult to identify particular a-subunits. It is suggested that purified or recombinant a-subunits be first resolved on these gels and used as reference standards. 29. Be careful. After blotting, the tank buffer is very hot, sometimes approaching 80-90°C 30. Appropriate antibody dilutions can be determined by immunoblotting different amounts of plasma membrane preparattons and selecting an amount that gives a strong mnnunoreactive response. 3 1 First and second antibody solutions can be stored at 4°C indefinitely, tf a small spatula amount of the antimicrobial agent thimerosol is added prior to each use. These solutions can be used three or four times. 32. If solubilizing membranes, take the appropriate amount, spin on a benchtop microcentrifuge for 5 min at top speed and remove supernatant. Resuspend membrane pellet in 100 pL H20 and add 100 pL 2% (w/v) SDS. Proceed with rest of the protocol. 33. Pansorbin is considerably less expensive than protein A-sepharose and should be considered as first choice. However, better results may be obtamed with protein A-sepharose. 34. Calbiochem Novabiochem (UK) (Freepost, Nottingham, UK) supply partially purified recombinant G protein a-subunits (including G,1,2,3, G, and GJ for use as standards on immunoblots.

177

G Protein a-Subunits Acknowledgments

We thank Dr. Morag A. Grassie and Dr. Alan Wise for discussion and useful suggestions in the preparation of this chapter.

References 1. Goldsmith, P., Gierschik, P., Milhgan, G., Unson, C. G., Vinitsky, R., Malech, H., and Spiegel, A. (1987) Antibodies directed against synthetic peptides distmguish between GTP-binding proteins in neutophil and bram. J Blol. Chem. 262, 14,683-14,688. 2. Wise, A. and Milligan, G. (1994) High level expression of mammalian G protein cxsubunit Gq subtypes in Eschenchia coli. Biochem. Sot. Trans. 22, 12s. 3. Koski, G. and Klee, W. A. (1981) Opiates inhibtt adenylate cyclase by strmulating GTP hydrolysis. Proc. Natl. Acad. SCL USA 78,4185-4189. 4. Hudson, T. H. and Johnson, G. L. (1980) Peptide mapping of adenylyl cyclase regulatory proteins that are cholera toxin substrates J. Blol Chem 255,7480-7486. 5. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-695. 6 Towbin,

H., Staehelin,

T , and Gordon,

J (1979)

Electrophoretic

transfer

of pro-

teins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Nat1 Acad. Sci. USA 76,4350-4354.

13 Covalent Modification of G Proteins by Affinity Labeling Martin Hohenegger,

Michael Freissmuth,

and Christian

Nanoff

1. introduction The basic mechanism of G protein-mediated transmembrane signaling was elucidated in the late 1970s and early 1980s. Subsequently, molecular clomng has identified a large array of closely related receptors (R), G protein subunits (G), and effecters (E). Currently, an important research goal is to understand the factors that, at each step of the cascade, link R to G to E, govern signal transfer, and signal integration following activation of a specific receptor (I). Receptors, in particular, appear to differ widely in their ability to interact with the different G protein oligomers and examples exist for stringent coupling of a receptor to a single species of a heterotrimer composed of a defined a-, p-, and y-subunit, as well as promrscuous interaction with a wide variety of different G protein subunits (2) Several experimental strategies have been employed to identify the G protein subunits that interact with a given receptor. These are dealt with m other chapters of this volume. Here, we will focus on methods for covalent labeling of G protein a-subumts, which can be used to study receptor G protein-coupling by exploiting receptor-promoted guanine nucleotide exchange. Two GTP analogs are well-suited for affimty labeling of the guanine nucleotide binding pocket, namely GTP-azidoanilide (=azidoanilido-GTP) and the 2’,3’-dialdehyde analogs of GTP and GTPyS (o-GTP, o-GTPyS). When bound to a G protein, GTP-azidoanilide cannot be hydrolyzed and therefore remains tightly associated with the protein. Covalent incorporatron is achieved by UV-photolysis. o-GTP and o-GTPyS form a Schiff s base with the s-amino group of lysine residues in the guanine nucleotide binding pocket of the protein; reduction with borohydride results in the formation of a covalent bond. Compared From

Methods III Molecular B/ology, vol 83. Receptor SIgnal Transduction Edited by R A J Chalks Humana Press Inc. Totowa, NJ

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Protocols

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with other affinity probes (e.g., 8-azido-GTP), these ligands offer the advantage of binding in a quasi-irreversible manner. Hence, the free ligand can be removed; this greatly reduces background labeling and improves the signal-tonoise ratio. 2. Materials The laboratory should be equipped for standard biochemical techniques involvmg radioactive reagents, i.e., possess a fanned hood reserved for handling radioactive substances,lead bricks, plexiglass shields, liquid scintrllation counter, slab gel electrophoresis, HPLC equipped with a gradient-forming pump system, fraction collector, and filtration manifold. 2.7. Synthesis and Purification of [a-32P]GTP-Azidoanilide 1. [Y-~~P]GTP1mCi, specific activity 3000 Ci/mmol (NEN). 2. 4-azidoaniline (Serva). 3. 1,Cdioxane (Merck). 4. N-(3-dimethylaminopropyl)-II$-ethylcarbodiimide hydrochloride (Merck). 5. 2-(N-morpholino)ethanesulfonic acid (Merck). 6. 1Mtriethylammonmm carbonate(=TEAC, Fluka). 7. Ethanol. 8 SupelcosilLC 308 (RP C18-5 um) HPLC cartridge (Supelco). 9. Neutral alumina (Merck). 10. Plexiglass shields, plexiglass shades,Geiger-Mtiller counter; HPLC and lyophilizer reserved and adaptedfor handling radioactivity in mCi quantities. 2.2. Synthesis and Purification of 2’,3’-Dialdehyde Analogs of GTP and GTPyS 1. Radioactively labeled [35S]GTPySand [a-32P]GTP(NEN). 2. Unlabeled guanine nucleotides:GTP, GTPyS, GDP (Boehrmger Mannheim or Sigma). Commercial preparations of GTPyS(and of GppNHp, another hydrolysis-resistantGTP analog) are rarely 100%pure, most often the contamination1s GDP; this canbe removedby chromatographyover anappropriately srzedDEAESephacel(Pharmacia)column. Briefly, pour a 20-tnL column, wash with 1M LiCl in Hz0 (30 mL), equihbrate in Hz0 (100 rnL); dissolve 50 mg GTP@ in 5 mL H20, apply onto column, wash with I5 mL HzO, elute guanine nucleotides with 80 mL gradient of 0-IMLiCl; collect 2-mL fractions; follow elution either with on-line UV-detector or by diluting samples 1: 100 in Hz0 and determining the absorbance at 252 mn in a UVNis-spectrophotometer (quartz cuvet); typically, two peaks will be detected, one at 0.4MLiC1, corresponding to GDP (-20%), and one at 0.7MLiC1, corresponding to GTPyS (-80%). Pool GTPyS containing fractions and dispense into 2-4 centrifuge tubes; add 9 parts of ice-cold ethanol, store

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overnight at -2OOC (lithium salts of nucleotides precipitate readily in ethanol); centrifuge, discard supernatant, lyophilize pellet to dryness, and dissolve in 5 mL Hz0 (adjust pH to 7-8 with NaOH), add dithiotbreitol to 2.5 mM final concentration; the concentration is best determined spectrophotometrically, using serial dilutions (1:200,1:400, and so on) and the molar extinction coefficient, E =I 3.7 m&P’/cm at 252 run. 3. NaBH4, NaCNBH,, and NaIO, from Merck (prepare fresh solutions immediately prior to use). 4. Glycerol (Fluka). 5. LiCl (Merck): Prepare 2M stock. 6. Dithiothreitol (DTT, Sigma): Prepare 1M stock; most GTPyS binding reaction buffers contain 1 mM DTT; we omit DTT in the current applications for several reasons: DTT can interfere with photoaffinity labeling by GTP-azidoanilide, with oxidation/reduction by NaI04/ NaBH4; DTT can activate some G protein-coupled receptors in the absence of agonists. 7. Sephadex G-10 (Pharmacia). 8, PEI-F cellulose (fluorescent-indicator coated) TLC plates (Merck).

2.3. Affinity Labeling of G Protein a-Subunits 1. Purified oligomeric

G proteins or G protein a-subunits; alternatively, appropri-

ate membranepreparationsor partially purified G proteins from native tissueare an adequate substrate. Membranes prepared from the cerebral cortex (gray mat-

ter) of mammalsare arich sourceof heterotrimeric Gproteins in which G, represents - 1% of the total membrane proteins. 2. HEPES/NaOH (pH 7.6): Make up as 1M stock solution. 3. Tris-HCl (pH 8.0): Make up as 1M stock solution. 4. EDTA make up as O.lM stock (adjust pH to 7.0 with NaOH; EDTA will dissolve as pH rises). 5. MgS04 and MgC12: Make up as 1M stocks. 6. NaCl: Make up as 4it4 stock. 7. Lubrol PX (Sigma): Make up as a 10% aqueous solution (w/v); add 5 g of mixed bed resin AG501-X8 (Bio-Rad) /l L of Lubrol solution and stir overnight at room

temperature;filter in themorning; dispenseinto ahquotsandstorefrozen at-20°C. 8. Binding reaction buffer: Make up 50 mL from stock solutions: 50 mM HEPES, pH 7.6, 1 mA4 EDTA, 10 mM MgS04, Lubrol O.l%, keep refrigerated.

9. Binding stop solution: Make up 2 L from stocks:20 mMTris-HCl, pH 8.0, 20 mM MgC12, 150 mA4 NaCl, keep refrigerated. 10. Filtration manifold (Hoefer).

11. BA85 nitrocellulose filters (Schleicher & Schuell) to trap purified G protein a-subunits in binding assays, or glass fiber filters (Whatman GF/C, GF/B) for membrane-bound proteins. 12. UV hand lamp (254 nm; 100-200 W).

13. Antisera for the identification of G protein o-subunits (seeChapter 12).

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3. Methods 3.1. Synthesis of [&*P]GTP-Azidoanilide The synthesis of [CL-32P]GTP-azidoanilide was originally described by Pfeuffer (3). The method outlined below is adapted from Offermans et al. (#), with some modifications. 1 [~z-~~P]GTP is freeze-dried in a plain glass tube and dissolved m 50 pL of 0.M 2-(N-morpholino)ethanesulfonic acid, pH 5.6, containing 1.5 mg of N-(3-dimethyl-aminopropyl)-M-ethylcarbodiimide hydrochloride. For most applications, the specific activity can be diluted to 300 Ci/mmol by the addition of 1.44 nmols carrier GTP. 2. 1,4-Dioxane (5 mL) is made peroxide-free by passing through a -1 -mL alumma column. 3. Incubate [cx-~~P]GTP with an excess of 4-azrdoaniline, i.e., 2 4 mg suspended (1 e., not dissolved) in 30 pL dioxane, for 4 h at room temperature Rotate the reaction tube slowly. During the entire procedure, the samples are best shielded from light. 4. [a-32P]GTP-azidoanilide is purified by HPLC on a supelcosil C,s reversed-phase cartridge. The HPLC is operated at room temperature with a flow of 1 mL/mm and the column IS equilibrated with 2 8% ethanol m 0. IMTEAC, pH 8.5. Instead of gassing triethylamme solutions with CO2 until neutral, we use the commercrally available (premade) TEAC as buffer Although the pH 1salkaline, this does not markedly reduce the efficiency of separation, but avoids installing bubble traps m the high pressure liqmd delivery system that would be required if carbonated buffer solutions were employed. 5. After application of the sample, the column is washed with eqmhbratron buffer (for 15 mm) to remove [cx-~~P]GTP (and contammating GDP), which appears typically at -5 mm after injection. [a-32P]GTP-azidoanllide is eluted by an ethanol gradient of 2 8-88% in 0 1M TEAC and 1-mL fractions are collected. The gradient is developed over a period of 18 min, followed by a IO-mm wash with 88% ethanol in TEAC. The desired reaction product appears 16-17 min after the start of the gradient. 6. Chromatographic resolution is satisfactory if the peak of radioactivity (containmg [a-32P]GTP-azrdoanilide) is separated from the following (within the tail of the radioactrvrty peak) fractions with a yellowish tinge. These samples contam aniline derivatives that interfere with [a-32P]GTP-azrdoanllide labeling; only poor labelmg of G protein a-subunits is obtained with this material. The color of the pertinent samples can be inspected in drm light after counting the radioactrvity m l-Ccs,aliquots. 7. About 75% of the radioactivity applied is incorporated into the GTP-azidoanilide. The clear, untinged radioacttvtty peak (typically over three 1-mL fractions) is combined and frozen in liquid nitrogen for lyophilization. Freeze-drymg of 3 mL takes -2 h. The dried sample 1s resuspended m 0.1 mL water, yrelding a concentration of - 1 pM at a specific activity of -3 pC+L.

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8. [a-32P]GTP-azidoanilide is kept m the dark and stored at -20°C. The stability of the compound is good and the shelf-life is limited by radioactive decay of the IX-~~P.

3.2. Synthesis of o-GTP and of o-GTPyS 1. For the synthesis of the unlabeled compounds, a typical reaction volume is 0.5-l 0 mL; make IO0 mA4 GTP (buffered to pH 7.0-8.0 wtth NaOH) and add 110 mMNaI04. 2. The reaction is carried out for 1 h on ice in the dark; stop by adding l/10 glycerol (final concentration 10% v/v). 3. Since the reaction products (iodate and aldehydes generated from glycerol) may interfere in subsequent measurements (in particular, nonspecific effects at high concentrations), a parallel mock synthesis is recommended. NaIO, is first reacted with glycerol, then GTP is added; obviously, this GTP remains GTP. If this solution behaves differently from authentic GTP in subsequent measurements, the observed effects are induced by reaction products. 4. Gel filtration: Gt,,-Sephadex (Pharmacia) is suspended m water and allowed to swell, and is then transferred into spin-columns (bed vol 1. l-l .6 mL). The column 1s prespun at -5OOg for 3 min in a swing-out rotor. 0.1-O. 15-n& aliquots (=lO% of bed volume after prespinning) of reaction mixture are added and the columns are spun again. If larger amounts of o-GTP are to be purified, a column (column dimensions =lO x 2 cm/l mL of 100 mA4 o-GTP) is poured m a cold room; equilibrated m water; the reaction mixture applied and fractions collected. Highly purified o-GTP is typically found in the ascending portion of the nucleotide peak. The elution should be monitored either with an on-line UV-momtor or by determining OD at 252 nm in a UV-spectrophotometer in individual fractions (1: 100 dilution of fractions; quartz cuvet) The concentration is determined as detailed m Section 2.2. 5. Yield: Typically, >95% of GTP and of GTpYS are converted to the 2’,3’-dialdehype species; the compounds are dispensed into aliquots and stored frozen at -80°C. We avoid repeated freezing and thawing and do not use stocks that are older than 1 mo. We have not systematrcally characterized the stability of the compounds, but prolonged storage of oxidized guanme nucleotides results in loss of biologrcal activity. 6 The synthesis of radtoactively labeled oxidized GTP analogs is carried out in a manner analogous to that described above for the unlabeled species; [a-32P]GTP or [35S]GTPyS are supplemented with unlabeled GTP or GTPyS to reach a final concentration of e 1 &and reacted with a 1.1 -fold molar excess of NaIO,. If the concentration of guamne nucleotides is reduced further, the rate of conversion of the parent compound to the oxidized species drops progressively. The unreacted compounds interfere with subsequent affimty labeling. We therefore do not recommend the sacrifice of purity for higher specific acttvity.

3.3. Chemical Identification

of the Products

The most rapid and convenientway to analyze the reaction products is by thin layer chromatographyon PEI-cellulose-F plates. The unlabeled guamne

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nucleotides are visualized with a UV lamp, radioactively labeled nucleotides by autoradiography. 1. GTP-azidoanilide: mobile phase: 0.2M Tris-HCl (pH 7.2), 1M LiCl. 1 pL each of HPLC purified [cr-32P]GTP-azidoanilide and of [a-32P]GTP are spotted onto the starting line of the TLC plate; 1 pL of unlabeled GTP and GDP (10 mM) each are also applied as reference. GTP-azidoanilide @f-OS) migrates slightly ahead of GDP (Rf -0.45), but the migration of the parent compound GTP IS slower (Rf-0.25). 2. o-GTP: mobile phase: lMLiC1, 0.5MTris (unbuffered); 1 pL each of 10 mM o-GTP and GTP are applied to the starting line on the plate. Prepare parallel sample, in which o-GTP is first reduced with NaBH4 (add lo-fold molar excess over o-GTP 15 min on ice). Visualize spots under UV-lamp: o-GTP does not leave the origin. After reduction with NaB&, o-GTP (Rf -0.8) migrates slightly ahead of GTP (Rf -0.75). GTP does not change its migration after incubation withNaB&. Rf-values for GTPyS and o-GTPyS are -0.56 and -0.65, respectively

3.4. Biochemical

Assays for the Synthesized

Several tests can be employed

GTP Analogs

to verify that the products of the synthesis are

indeed functionally active and thus useful for subsequent experiments. Depending on whether radioactively labeled or unlabeled compounds are to be tested, the following approaches can be used: 1. Direct incorporation of the radioactively labeled compounds into purified G proteins or membrane-bound G proteins. 2. Competition of the unlabeled compounds for bindmg of [35S]GTPyS to purified G proteins or membrane-bound G proteins.

Several additional straightforward methods can be used; two examples are listed in Note 1. We perform binding to purified recombinant G,a as a standard procedure to assessthe functional integrity of GTP-analogs; G,a exchanges prebound GDP rapidly and its kinetics for guanine nucleotide binding and hydrolysis, as well as its interaction with receptors, are well characterized (.5,6), but other well characterized a-subunits, such as G,a or appropriate membrane preparations, can be used. 3.4.1. Competition of Unlabeled GTP Analogs with rsS]GTPyS Binding GTP-azidoanilide, oxidized GTP analogs, and the radioligand [35S]GTPyS bind to G protein a-subunits in a quasi-irreversible manner; in contrast, GTP is hydrolyzed to GDP when bound to the protein and released again; the apparent affinity of GTP thus depends on the incubation time and decreases progressively with increasing incubation times. Hence, a second parallel incubation can be set up and incubated for four times the length of time (e.g., 2 h). The

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potency of GTP will decrease while that of GTP-azidoanilide and oxidized GTP analogs is not altered (7). 1. Use polypropylene tubes (3 mL) to minimize adsorptive losses of protein. Shield GTP-azidoanilide containing solutions from light. 2. Prepare an incubation medium (30 pL/reaction) containing binding buffer: 50 mM HEPES, pH 8.0, 1 mM EDTA, 10 mM MgS04, Lubrol 0.1% and 1.67 p&f [35S]GTI?@ (specific activity 10-20 cpm/fmol); check total activity by scintillation counting (0.5-l x lo6 cpm); maintain on ice. 3. Prepare a serial dilution of guanine nucleotides covering the range of 0.3-l 00 @4 as fivefold concentrated working solutions in binding buffer; use serial dilutions of GTP and/or GTPyS as a control; maintain on ice 4. Dilute GSo (or G,cx) in binding buffer to obtain -2 pmol of a-subunit/l0 pL; maintain on ice. 5. [35S]GTPyS is assayed in duplicate in a 50-pL incubation. Combine 30 pL of [35S]GTPyS-containing medium (step 2) with 10 pL buffer (control binding) or increasing concentrations of guanine nucleotides (see step 3) and 10 pL G protein solution (see step 4). The filter blank is determined in the absence of G protein. 6. Incubate the control binding reactions for 30 min at 2O”C, for the remaining tubes, the binding reaction is allowed to proceed for 2 h. The reaction is stopped by the addition of 2 mL stop solution (20 mMTris-HCl, pH 8.0,20 mA4MgClz, 150 m&f NaCl, 0.1 mM GTP). 7. Pour diluted reaction mixture over BA85 nitrocellulose filters and rinse filters with 20 mL of wash buffer (20 mMTris, pH 8.0,20 rnA4MgCl,, 150 mMNaC1); allow filters to dry, place in scintillation vials, add scintillation cocktail, and the radioactivity determined.

Under these assay conditions, i.e., with purified G proteins, the nonspecific binding corresponds to the filter blank and is generally very low if the filters are rinsed thoroughly (~100 cpm/105 cpm added). An analogous experiment can also be set up with membranes as the source of G proteins; in this case, detergents are to be omitted from the reaction. Glass-fiber filters and an semi automatic filtration apparatus (Skatron or Brandel) can be used to trap the protein-bound radioactive ligand. 3.4.2. Binding and Affinity Labeling of Purified G Proteins with [cx-32PIG TP-Azidoanilide Binding of [a-32P]GTP-azidoanilide to G,a (or G,a) is assayed as outlined under Section 3.4.1.) with the following modifications: 1. Prepare duplicate incubations in 25 pL containing 50 WHEPES, pH 8.0,l mM EDTA, 10 mMMgSO.,, Lubrol 0.1%; [o-32P]GTP-azidoanilide at a final concentration of 0.1-0.2 l,&f and a molar excess of G,a (e.g., 5 pmohassay). 2. To determine nonspecific incorporation, a parallel incubation is set up that contains 100 @funlabeled GTPyS. The assay tubes are wrapped in ahuninium foil.

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3 After an incubation of 1 h at 2O”C, aliquots (5 pL) are withdrawn, spotted onto a metal plate covered with a double layer of parafilm (press the conical end of an Eppendorf tube firmly onto the parafilm to create a well from which the sample can be easily retrieved); irradiate for 0.5 min with UV-hght from a distance of 3 cm. These samples are subjected to SDS-polyacrylamide gel electrophoresis, the labeled bands identified by autoradiography of the dried gel, excised, and counted for radioactivity 4 The remaining mcubations are diluted by the addition of 2 mL ice-cold stop solution (20 mM Tris, pH 8.0, 20 mA4 MgCl,, 150 mM NaCI, 0.1 mM GTP) and filtered over BA85-nitrocellulose filters (see step 7 of Section 3.4.1.). 5. The filters are rinsed with 20 mL of stop solution, dried, taken up m liquid scmtillation cocktail, and the radioactivity is determmed. Since an excess of G protein a-subunit over GTP-azidoanilide is present in the incubation, essentially all of the radioactivity ought to be bound to the protein, as determined by filter binding. However, since the nitrene generated by UV-madration can also react with water, the covalently incorporated amount of radioactivity is generally much lower. Using the comparrson outlined above, we have found that -5% of the bound radioactivrty is indeed incorporated into the protein.

3.4.3. Affinity Labeling of G Proteins with [cx-~~P]o-GTP and f5S]o-GTPyS 1. G,a (10 pmol) is incubated in 25 pL buffer (50 mMHEPES, pH 7.6, 1 mMEDTA, 10 mM MgS04, Lubrol 0.1%) contaming 10 ~ [cx-~~P]o-GTP or [35S]o-GTPyS (specific activity 20 cpm/fmol) 2 After an incubation period of 1 h at 2O”C, add 5 pL of a freshly-prepared aqueous solution of NaBH4, to reach a final concentration of 0.1-l mA4 (minimum lo-fold excess over o-GTPlo-GTPyS); the reduction is carried out for 1 h on me. 3. Add Laemmli sample buffer containing 40 mMDTT, denature by heating ( 10 min at SOY!), and apply to SDS-polyacrylamide gel; the labeled band is identified by autoradiography of the drted gel, excised, and counted for radioactivity. Expect a labeling stoichiometry of -0.5.

3.5. Affinity

Labeling

by In Situ Oxidation-Reduction

Membrane-bound GTP-binding proteins, GTP-binding proteins in cellular extracts, and purified G proteins can be affinity labeled after they have been allowed to bind radioactively labeled GTP or GTPyS (7-9). In most G proteins, the drssocration of GTPyS approaches zero m the presence of millimolar free magnesium concentrations, while GTP is hydrolyzed and the GDP formed 1s released. Hence, [35S]GTPyS is recommended for affinity labeling. For purified G proteins, the labeling reaction is a logical extension of the GTPyS-bind-

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ing assay. We will therefore only outline the method used to label membranebound G proteins. 1. Prepare reaction buffer (20 mA4 HEPES/NaOH, pH 7.6, 1 mM EDTA, 2 mM MgS04; alternatively, tf Na+ is to be avoided, prepare 20 mM Tris-HCl, pH 7.6, 1 miVEDTA, 2 mM MgClz) Make up binding buffer (50 &/assay) by adding [35S]GTPyS (1 x lo6 cpm) and the desired concentration of unlabeled GTPyS (O-2 I.& final concentration); check total activity by scintillation counting and use to calculate specific activity; hold on ice. The choice of free magnesium concentration, final GTPyS concentration, incubation times, and supplementation with GDP are critical, tf the receptor-catalyzed exchange is to be momtored (see Note 2). 2. Weigh out required amount of NaI04, NaCNBH3, and NaBH4 to obtain 10 x 4 mA4,2 x 80 mM, and 10 x 10 rr&Z in HZ0 Do not yet add water. 3. Prepare 20 mM Mg buffer (= reaction buffer containing 20 mM MgC12) and GTPstop buffer (= 0.1 mMGTP m 20 mMMg buffer; 1 ml/assay); mamtam both on ice. 4. Dilute membranes into reaction buffer to yield 100 ug/50 pL (see Note 2, for an estimate of the required amount); maintam on ice. 5. Combine membrane dilution (50 pL) and [35S]GTPyS-contammg binding buffer (50 pL) in Eppendorf tubes. If receptor agonists/antagonists are to be evaluated, combine appropriate dilutions first with membranes. For short incubations, prewarm both the membrane suspension and the binding buffer to desired temperature (20-3O’C). 6. Incubate for desired period; add ice-cold 1 mL GTP stop-buffer (see step 3) to stop the binding reaction; spin in refrigerated centrifuge for 20 min at 50,OOOg; toward the end of the centrifuge run, dissolve preweighed NaI04, NaCNBH3, NaBH4 in water. 7. Quickly withdraw supernatant and resuspend pellet in 0.1 mL Mg buffer (see step 3) by rapidly ptpeting the solution up and down or by briefly immersing the tubes in a sonicating waterbath filled with me-cold water. 8. Add 11 I.~Lof NaI04 (4 rnA4 final); incubate for 1 min at 20°C. 9. Add 110 & NaCNBH3 (80 mA4 final); incubate for 1 min at 20°C. 10. Add 22 pL of NaBH4 (10 mM final); incubate for 30 min at O’C 11. Centrifuge (Eppendorf microcentrifuge maximum speed), withdraw supematant, dissolve pellet in Laemmli sample buffer contaimng 40 miV DTT and 1% SDS, heat denature and apply to SDS-polyacrylamide gel. Identify labeled bands by autoradiography. Alternatively, dissolve membrane in appropriate buffer for immunoprecipitation with specific antisera (see Chapter 12).

3.6. Affinity Labeling of Membrane with [a-32P]GTP-Azidoanilide

Bound G Proteins

The labeling reaction in membranes is carried out by following steps 1 and 3-5 in Section 3.5. Handling of the photoaffinity probe [a-32P]GTP-azidoanilide is as according to Section 3.4.2. (steps 2-5).

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4. Notes 1. Synthesis: We recommend that the synthesis be carried out first with unlabeled GTP or GTPyS. This allows the investigator to become familiar with the procedure without the additional stress of handling mCi quantities of radioactivity. Similar to GTPyS (and GppNHp), o-GTPyS and GTP-azidoanilide are not hydrolyzed and thus irreversibly activate G protein a-subunits and their effecters; this feature therefore can also be exploited to verify that the synthesis and purification yields active compounds. For instance, membrane preparations can be used to compare the activation of adenylyl cyclase in the presence of o-GTPyS and GTP-azidoanilide, with the stimulation induced by GTPyS (3,7). Similarly, guanine nucleotides destabilize high-affmity binding of most agonist radioligands to G protein-coupled receptors m membrane preparations (see ref. IO for an example). 2. Receptor-dependent activation: The goal of most researchers is to determine which G protein a-subunit is activated by the receptor under investigation in a membrane preparation obtained from cells or tissues. This can be achieved in a seemingly straightforward manner by determining if the receptor Increases the covalent incorporation of GTP analogs into G protein a-subunits. However, it is important to design the experiment appropriately and the following points ought to be considered: a. How abundant are the candidate G protems? Typically G, and G, are expressed at higher levels than G protems of the G,, G, and Glzj,s-classes. The expression level determines how much membrane protein is to be used in the incubation and how high the specific activity of the radioactively labeled GTP analog is required to be to obtain detectable labeling. G protein levels can be estimated by immunoblotting with appropriate antisera. It is wise to use appropriate standards (purified proteins or, alternatively, membrane preparations, which have previously been characterized with respect to their expression levels). b. How fast do the G proteins exchange guanine nucleotides spontaneously, i.e., without activation by an agonist-activated receptor? The rate at which G protein a-subunits release prebound GDP differs widely, even among closely related proteins; e.g., G,a exchanges very rapidly, but the retinal G protein transducin (G,a) has an extremely slow rate of exchange. The rate of exchange in the heterotrimeric G protein is also Influenced by the magnesmm concentration (which accelerates GDP release) and the NaCl concentration (which decreases GDP release). The agonist-activated receptor induces a conformational change so that GTP and GTP analogs are bound preferentially, even in the presence of GDP. Finally, once bound hydrolysis-resistant GTP-analogs are bound quasi-irreversibly as long as the magnesmm concentration is kept m the millimolar range. c. When does the receptor-induced activation reach its maximum? During membrane preparation, receptors and G proteins are physically separated. The ratio between receptor and G protein, which are in the same vesicle, can be assumed to approximate a random distribution. In general, G proteins are present m

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excess of the receptor density; some vesicles will therefore contain G proteins and no receptor, but the reverse will be a less likely event. An activated receptor in a vesicle will rapidly exhaust the supply of accessible G proteins. Thus, long incubation periods favor the detection of low basal guanine nucleotide exchange. d. If reasonable guesses are not available for the system under mvestigation, preliminary expertments are recommended in which agonist-dependent [35S]GTPyS binding is determined in membranes; the optimal signal-to-noise ratio (i.e., a large agonist-induced increment of [35S]GTPyS-binding) can be assessed from the time-course of activation as well as from the effect of GDP, Mg2+, and NaCl (see Chapter 8); combined with the concentration of receptor derived from binding assays with an appropriate radioligand, the molar turnover number of the receptor can be estimated, i.e., the number of G protein molecules activated per receptor (R) m a given time interval (mol GTPyS bound/mol R/mm). 3. Oxidized GTP analogs vs GTP-azidoanilide: Whether oxidized GTP analogs or GTP-azidoamlide are employed depends on the application. For the analysis of receptor-dependent activation of G protems, [a-32P]GTP-azidoanilide has been widely used, since it can be obtained at very high specific activity. However, not all G proteins bind GTP-azidoanilide readily (I 1). The method of in situ oxidation-reduction with [35S]GTPyS lends itself to the identification of unknown GTPbinding proteins. In addition, under appropriate conditions, unlabeled o-GTP can be used to irreversibly block endogenous GTP-binding proteins; the disrupted signaling pathway may subsequently be reconstituted by the exogenous addition of purified G proteins (12).

References 1. Milligan, G. (1993) Mechanisms of multifunctional signalling by G protein-lmked receptors. Trends Pharmacol Sci. 14,239-244. 2. Offermanns, S. and Schultz, G. (1994) Complex information processing by the transmembrane signalmg system involving G proteins. Naunyn Schmiedeberg ‘s Arch Pharmacol. 350,329-338. 3. Pfeuffer, T. (1977) GTP-binding proteins in membranes and the control of adenylate cyclase activity. J. Biol Chem. 252,7224-7234. 4. Offermanns, S., Schultz, G., and Rosenthal, W. (1991). Identification of receptoractivated G proteins with photoreactive GTP analog, [a-32P]GTP-azidoanilide. Methods Enzymol. 195,286-302. 5. Graziano, M. P., Freissmuth, M., and Gilman, A. G. (1989) Expression of G,o? in E. coli: purification and characterization of two forms of the protein. J. Bioi Chem. 264,409-4 18. 6. Freissmuth M., Selzer E., Marullo S., Schhtz W., and Strosberg, A. D. (1991) Expression of two human a-adrenergic receptors m E. ~011: functional interaction with two forms of the stimulatory G protein. Proc. Natl. Acad. Sci. USA 88, 8548-8552.

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7. Hohenegger, M., Nanoff, C., Ahom, H., and Freissmuth, M. (1994) Structural and functional characterizatron of the interaction between 2’,3’-dialdehyde guanme nucleotide analogues and the stimulatory G protein a-subunit. J. Bzol Chem. 269, 32,008-32,0 15. 8. Low, A., Faulhammer, H G., and Sprinzl, M (1992) Affinity labeling of GTPbinding proteins in cellular extracts FEBS Lett. 303, 64-68. 9. Hohenegger, M., Mitterauer, T., Voss, T., Nanoff, C , and Frerssmuth, M. (1996) Throphosphorylation of the G protein a-subunit in membranes: evidence against a direct phosphate transfer reaction to Ga-subunits. Mol. Pharmacol. 49,73-80 10. Jockers R., Linder, M. E , Hohenegger, M., Nanoff, C., Bertm, B., Strosberg, A. D., Marullo, S., and Fretssmuth, M. (1994) Species dtfference m the G protein selectivity of the human and the bovine A,-adenosme receptor. J. B~ol Chem 269, 32,077-32,084. 11. Fields, T. A., Lmder, M. E., and Casey, P. J. (1994) Subtype-specrfic binding of aztdoamlido-GTP by purified G protein a-subunits. Bzochemistry 33,6877-6883. 12 Nanoff, C., Boehm, S., Hohenegger, M., Schutz, W , and Freissmuth, M. (1994) 2’,3’-dialdehyde-GTP as an irreversible G protein antagonist. J Biol Chem. 269, 3 1,999-32,007.

14 Heterologous Expression of Receptors and Signaling Proteins in Adult Mammalian Sympathetic Neurons by Microinjection Stephen R. lkeda 1, Introduction Heterologous expression of receptor proteins provides a means of studying a molecularly defined receptor subtype in isolation from species closely related either by function and/or homology. For example, metabotropic glutamate receptors (mGluRs) comprise a large family (presently eight distinct subtypes, plus splice variants) of G protein-coupled receptors (GPCRs) for which L-glutamate is the endogenous hgand (1,2). However, identifying a response arising from a single natively expressed mGluR subtype is problematic, because pharmacological agents with the required specificity are not currently available. Moreover, other proteins that interact with L-glutamate, such as ionotropic glutamate receptors (11 or glutamate transporters, are often coexpressed m neurons along with mGluRs, thus confounding the situation further. Finally, even the apparent presence of only a single receptor subtype in a given cell (e.g., as determined by mnnunohistochemistry) is not sufficient to allow unambiguous assignment of a response to a particular receptor subtype, since additional uncharacterized members of a receptor family may exist. Thus, assigning roles to, and characterizing signaling pathways of, specific mGluR subtypes m their native environment is a difficult task. The problem of receptor identity can be largely alleviated if receptors are heterologously expressed in cells normally devoid of a measurable response to agonist application (i.e., a null background). Two systems commonly used for the heterologous expression of GPCRs are the Xenopus oocyte and clonal mammalian cells (e.g., COS, CHO, HEK293, and so on). In Xenopus oocytes, From

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GPCRs are usually expressedby mlcroinjectmg in vitro transcribed complementary RNA (cRNA) into the cytoplasm of the oocyte. In contrast, GPCR expression in mammalian cell lines is accomplished by transient or stable transfection of complementary DNA (cDNA) using a variety of standardmethods (calcium phosphate precipitation, lipofection, electroporation, and so on). In both systems, responses resulting from the activation of GPCRs are detected from effector elements (e.g., Ca2+-dependent Cl- channels in oocytes and adenylyl cyclase in mammalian cells) already present in the expression host. The situation becomes more complicated when the effector molecules to be studied, in our caseneuronal voltage-gated ion channels,are not present in the host cell. Although one can attempt to coexpressboth the GPCR and ion channel, this approach can be difficult for several reasons.First, some ion channelshave not yet been cloned (e.g., M-type IS+-channels)and thus heterologous expression is not feasible. Second, many ion channels are heteromultimers and thus require the assemblyof multiple subunits (e.g.,Ca2+channels)for functional activity (3). Moreover, knowledge of the precise subunit composition and stoichiometry of voltagegated ion channels is still evolving and thus the effect of these variables on modulation is unknown. Finally, some signaling elements that may be important for modulation of neuronal ion channel, such as the G protein subtype GO(#), are often absent from the nonneuronal cells commonly used as expression hosts (5). To circumvent these problems, we have developed an expression strategy, whereby GPCRs are heterologously expressed in adult mammalian sympathetic neurons by either cytoplasmic injection of cRNA or intrapuclear injection of cDNA. The heterologously expressed receptors readily couple via endogenous G proteins to natively expressed K+- and Ca2+-channels, thus allowing the detailed examination of molecularly defined receptors subtypes in a neuronal environment (6), In addition, we have recently extended this strategy to include expression of transduction elements and have used this methodology to identify the G protein subunit that mediates N-type Ca2+channel modulation in sympathetic neurons (7). Finally, preliminary experiments indicate that a variety of other proteins can also be expressedin sympatheticneurons and potentially used as biosensors to monitor intracellular events. Here is described the preparation of dissociated adult rat superior cervical ganglion (SCG) neurons and methods for expressing heterologous proteins in these neurons by cytoplasmic and nuclear microinjection of cRNA and cDNA, respectively. 2. Materials 2.1. Neuron Dissociation and Short-Term Culture 1. Hanks’ Balanced Salt Solution (HBSS): Prepare from 1 L powder mix (Life Technologies, Gatthersburg,MD) per manufacturer’s instructions. Store at 4°C in a 1-L bottle

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2. Modified Earle’s Balanced Salt Solution (mEBSS): Prepare 100 mL by adding 10 mL of 10X concentrated hquid EBSS (cat. no. E 7510, Sigma, St. Louis, MO) to about 70 mL of deionized water. Add 1.0 mL of 1M HEPES (cat. no. H 0887, Sigma), 0.36 g glucose, and pH the solution to 7.4 with 1NNaOH. Add 0.220 g NaHCO, and bring up the volume to 100 mL in a volumetric flask with deionized water Sterilize the solution by passing through a 0.2~pm filter and store lo-mL aliquots in tightly sealed 15-mL sterile polypropylene centrifuge tubes at 4’C. 3. Enzyme solution: Prepare enzyme solution just prior to the incubation step (see below). To 10 mL of mEBBS (brought to room temperature), add 7 mg of collagenase D (cat. no 115932, Boehringer Mannheim, Indianapolis, IN), 3.5 mg trypsin (cat no 109827, Boehringer Mannheim, see Note 2), and 1 mg DNase I, Type II (cat. no. D5025, Sigma). Invert the centrifuge tube gently (to avoid foaming) several times to ensure that the enzymes are properly dissolved. The concentrations of collagenase and trypsm are critically dependent on specific lots of enzyme and thus must be determined empirically for each new lot (see Note 1). 4. Culture media: Mimmal essential media (MEM) with Earle’s salts supplemented with 1% glutamine (200 mM stock), 1% penicillin-streptomycm solution, and 10% fetal calf serum (all from Life Technologies). 5. Poly-L-lysine coated 35-mm dishes: Dissolve 10 mg poly+lysme (cat. no. P 1274, Sigma) in 100 mL of O.lMsodium borate buffer, pH 8.4 (with HCl), and pass the solution through a 0.2~pm filter. Although the borate buffer is stable for long periods of time, we prepare the poly+lysine solution just prior to use. Add 1 mL of solution to each 35-mm dish and allow to stand (usually in the culture hood) for several hours (overnight is convenient). Aspirate off the poly-L-lysine solution and rinse the dishes twice with sterile distilled water. After the dishes are dry, they can be stored mdefimtely. We place six to eight 35-mm dishes in a 150 x 25-mm tissue culture dish to facilitate handling. 6. TE buffer: 10 mMTris-HCl, 1-d EDTA, pH 8.0 7. Equipment: dissecting microscope (Model SMZ-2T, Nikon) with light source, Dumont #5 tine forceps (Fine Science Tools, Foster City, CA), micro sprmg scissors (cat. no. 15024- 10, Fme Science Tools), a centrifuge capable of maintaming a low speed (500 rpm, Model HN-SII, International Equipment, Needham Heights, MA), tissue culture incubator, and a laminar flow culture hood (optional).

2.2. Microinjection 1. 0.1% Fluorescein dextran solution: Weigh out about 1 mg of 10,000 MW fluorescein dextran (cat. no. D 182 1, Molecular Probes, Eugene, OR) in an autoclaved 1.5-mL microcentrifuge tube and then add molecular biology grade Hz0 to a final concentration of 1 mg/mL. Filter the solution through a 0.2~pm syrmge filter and store m 100~pL aliquots in mtcrocentrtfuge tubes at 4°C. Since this solution is used with RNA, standard precautions should be taken to prevent RNase contamination. Thus, forceps used to handle the fluorescein dextran are wrapped in aluminum foil and autoclaved or baked (350°C for 2-3 h), all

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plasticware (pipet tips and centrifuge tubes) is autoclaved, and gloves are worn while preparing or handling the solution. Microinjection equipment: Our micromJection station (which is also used as a patchclamp setup) consists of a Nikon Diaphot-TMD inverted microscope equipped with 20 and 40x phase contrast objectives, an epifluorescence unit consistmg of 100 W Hg lamp illummation source, and a Nikon B2A filter cube (excitation filter 450-490 nm, dichroic mirror 510 nm, emisston filter 520 nm), a remote head CCD camera (Model 6410, Cohu, San Diego, CA), a g-in. black and white video monitor (Model PVM-122, Sony, Tokyo, Japan), an automated mtcromjectton system consistmg of an Eppendorf (Madison, WI) 5242 microinjection unit and 5 17 1 micromanipulator system. The microscope and associated equtpment are mounted on a vibration isolation table (Techmcal Manufacturing, Peabody, MA). Microinjection pipets: Two options are available. Commercially prepared injection micropipets can be purchased (Femtotips; Eppendorf) that are convenient, sterile, and RNase free. However, the plpets are expensive (about $6-7 each) and the tip geometry is fixed by the manufacturer. Alternatively, mtcropipets for inJection can be manufactured in the laboratory (see Note 6). Microcentrifuge tubes made from hematocrit capillaries: Plain (i.e , unheparmized) glass hematocrit capillaries are cleaned with consecuttve washes in acetone, methanol, and deionized H,O, and then dried m an oven. With some brands of tubes, the blue paint stripe must first be removed from each tube with an acetone soaked Kimwipe. The tubes are then fire-polished shut on one end with a Bunsen burner and placed in a clean glass beaker. The beaker is covered with aluminum foil and placed in an oven at 350°C for several hours to destroy RNase activity Hematocrit centrifuge (Marathon 13K/H, Fisher Scientific, Pittsburgh, PA).

3. Methods 3.1. Preparation of Dissociated Neurons 3.1.1. Dissection of the Rat Superior Cervical Ganglion 1. Adult male Wistar rats weighing 250-350 g are decapitated with a laboratory guillotine. The animal can be anesthetized prior to sacrifice if this will not interfere with the experiment. 2 The head is first placed in a lOO-mL plastic beaker containing cold HBSS to rinse blood from the dissection area. Subsequently, the head is placed ventral side up on in a dissecting plate, a 1SO-mm plastic culture dish is tilled one-half full with Sylgard 184 silicone elastomer (World Precision Instruments, Sarasota, FL), and a midline longttudinal incision is made in the ventral neck skin with scissors. The skin is then retracted laterally and pinned to the bottom of the dissectmg plate, thereby exposing the neck musculature. 3. Under a dissecting microscope, the common carotid artery is identified bilaterally and followed cranially until the carotid bifurcation is encountered. The superior cervical ganglion (SCG) lies on the medial side of the carotid brfurcation, between the internal and external carotid arteries (see Note 3). Both carotid bifur-

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cations, with SCG attached, are carefully dissected from surrounding tissue and placed in a 60-mm culture dish containing cold (4’C) HBSS. During removal of neck musculature to expose the SCG, flush the dissection field often with cold HBSS from a Pasteur pipet to clear blood, remove K+ released from damaged muscle, and to cool the dissection area. Further dissection is camed out with the carotid bifurcation (which IS Y-shaped) secured, with the SCG facing upwards, to the bottom of a small dissecting plate (60~mm culture dish half-filled with Sylgard) with 100~pm insect pins (Carolina Biological Supply, Burlington, NC) During the dissection, the preparation is covered with cold HBSS, which can be periodically replaced to remove debris and keep the tissue cool. Under a dissecting microscope, a longitudinal slit is made with fine spring scissors in the connective tissue capsule enclosing the ganglion. The ganglion 1sthen teased out of the connective tissue capsule, using blunt dissection with #5 Dumont forceps. Nerves entering and leaving the ganglion (usually three major ones) are cut close to the ganglion body with spring scissors. The desheathed ganglion is then transferred, using a large-bore fire-polished Pasteur pipet, to a 60-mm culture dish filled with cold HBSS and sitting on ice. When both ganglia have been desheathed, multiple parallel slits about 1 mm apart are made perpendicular to the long axis of the ganglia, using spring scissors to facilitate enzyme penetration. The free-floating ganglia can usually be gently pushed into the scissors blades with the tips of the Dumont forceps, without actually grabbing the tissue. We prefer to make slits about 75% of the way through the ganglia, which facilitates the transfer of the tissue as one piece. However, equivalent results are produced by cutting the ganglia into small pieces (about 1 mm3) using small scalpel blades The procedure is done using the dissecting microscope at fairly high magnification (x50). At this point, the ganglia are ready for enzyme dissociation. Although the dissection should be carried out expeditiously, peripheral neurons seem quite hardy as long as they are bathed in cold (4’C) HBSS; thus haste, at the expense of gentle handling of the tissue, does not seem warranted.

3.1.2. Enzymatic Dissociation and Culture 1. Enzyme solution (10 mL) is prepared as described (Section 2.1 , item 3) Six milliliters of the enzyme solution is pushed through a 0.2~pm syringe filter (Millex-GV, Millipore, Bedford, MA) into a 25-cm2 tissue culture flask with a plug seal cap (Falcon #3013). The ganglia (see Section 3.1 .l., step 7) are then transferred to the flask with a fire-polished Pasteur pipet, taking care to transfer as little HBSS to the enzyme solution as possible. 2. The flask is flushed with a mixture of 95% 02/5% COZ for -1 min and the cap tightly sealed. 3. Place the flask in a 35°C shaking water bath with the long axis of the flask aligned parallel to the stroke direction. The flask is shaken fairly rapidly (240 strokes/ min), but not so rapidly as to cause solution to slosh up the sides of the flask

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4. After 1 h, remove the flask from the water bath. At this point, the gangha should be m small separate pieces about 1 mm3 The pieces are then dispersed into single neurons by grasping the neck of the flask between the thumb and first finger and vigorously shaking the flask for 10 s with a motion similar to that used to shake the Hg down into the bulb of a rectal thermometer (the old-fashioned kind, not the new electronic types). 5. Following the shake, the pieces should be completely dispersed, with at most one or two barely visible pieces. 6. Five milliliters of culture media (preheated to 37°C) are added to the flask and the contents transferred to a 15-mL centrifuge tube 7. The solution of dispersed neurons is centrifuged for 5 min at 5Og in a swingmg bucket centrifuge at room temperature, the supernatant removed, 10 mL of culture media added to resuspend the small pellet, and the centrifngation repeated. 8. Following removal of the supematant, the pellet (which may be barely visible) is gently resuspended with 1.8 mL of culture media. 9. Neurons are plated into six 35-mm poly+-lysine coated tissue culture plates (Coming #25000). To keep the neurons centered in the dish (which facihtates injecting), an autoclaved glass cloning ring (cat. no. 2090-01010, Bellco Glass, Vineland, NJ), treated with Sigmacote (Sigma), is placed m the center of each 35-mm dish, and 0.3 mL of the dispersed neuron solution placed carefully in the cloning ring Two milliliters of culture media is then carefully added to the 35-mm culture dish, taking care not dislodge the cloning rmg. 10. After 5-6 min (which allows the neurons to settle), the cloning rings are carefully removed from the dishes with sterile forceps and the dishes gently moved to a tissue culture incubator (37”C, humidified atmosphere of 5% CO2 in air). 11. Immediately after dissociation, the neurons usually have several small stumps of amputated processes and are loosely attached to the substrate of the dish However, after 2-3 h of incubation, the neurons resorb the processes, attain a spherical geometry, and attach firmly to the dish. 12. Neuron somata are usually 20-40 pm in diameter, with a large nucleus containing 2-3 nucleoli

3.2. Injection

of cRNA into the Cytoplasm

of Neurons

1. In all procedures involving RNA, precautions are taken to avoid RNase contamination. Thus, gloves are worn while handling the sample, all plasticware (microcentrifuge tubes and pipet tips) that comes in contact with the sample is autoclaved, glassware is baked (350°C for several hours), and solutions are made from molecular biology grade or DEPC-treated HzO. 2. In vitro transcribed, capped cRNA coding for the desired protein is stored at -BOY, usually at a concentration of l-5 g/pL in Hz0 (see Note 9). 3. Mix l-3 pL of cRNA solution with 2-4 pL of 0.1% fluorescein dextran solution We find that a final concentration of 1 pg/$ is a good starting point. The small volumes can be mixed on a piece of Parafilm, while being observed with a dissetting microscope. Stick a small square of Paratilm to a surface with a drop of

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water (uncovered side down). Carefully remove the paper backing and use the resulting clean surface for mixing the drops of solution by plpetmg up and down several times with a plpetor. Transfer the 5 $ of solution to a specially prepared sealed-off hematocrit tube (see Section 2.2., item 4) with an autoclaved microloader pipet tip (cat. no. 5242 956.003, Eppendorf) The solution will usually be m the middle of the tube (i.e., not at the bottom). Centrifuge the tube for 10-20 min at 16,000g in a hematocrit centrifuge at room temperature. Place the hematocrit tube on ice. We use a 1 x 1 x 6 in. brass bar with holes drilled in it to hold the hematocrit tubes. The bar is placed in a small Styrofoam container filled with ice, which serves as both a holder and heat sink. Cut the hematocrit tube 3-4 cm from the bottom by scoring with a diamond pen. This allows the microloader tip to reach the solution. Pipet 1 & of cRNA solution into a microloader tip, being careful to remove the solution from the top of the fluid column. Avoid agitating the solution near the bottom of the tube, since this invanably stirs up particulate matter that then clogs the submlcron opening of the microinjection plpet. Backfill the injection micropipet with the 1 pL of cRNA solution by deposltmg the solution into the shank of the pipet. The glass capillary in the plpet will cause the remainder of the micropipet tip to fill by capillary action. Sometimes a small bubble forms, but it can be dislodged by gently flicking the micropipet wltb a finger. Place a dish of dissociated neurons (3-6 h after isolation) on the microscope stage, attach the injection micropipet to the holder, and position the micropipet about 100 pm above the bottom of the dish, using the 5 17 1 micromanipulator. We typically use a 20x phase contrast objective to view the neurons. The image is detected with a CCD video camera attached to the microscope and displayed on a video monitor. Although video equipment is not required, the inherently high contrast of the black and white video system enhances the phase contrast. Moreover, viewing the monitor is more comfortable than looking through the eyepieces during protracted injection sessions. When the injection micropipet is lowered into the culture media, the phase contrast image can be greatly degraded as a result of the meniscus formed at the glass-fluid interface. The image can be rapidly improved by slightly rotating the turret holding the phase rings one way or the other Gust off the “detent” position) to realign the phase rings. Cytoplasmic injections are carried out using the axial injection mode of the 5 17 1 micromanipulator at the default injection velocity (300 pm/s). InjectIon pressure (P2) usually ranges from 100 to 200 hPa and injection duration from 0.3 to 0.5 s, depending on pipet size. Holding pressure (P3) is set to about 45 hPa (not critical). The axial-injection z-axis limit is set with the following procedure. First, the micropipet tip IS positioned as close to the bottom of the dish as possible, using the step mode of the manipulator. This is facilitated by focusing on one of the very flat fibroblast-like cells (Schwann cells) that accompany the neu-

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rons in most preparations. The coordinates of the micromampulator are reset to zero, the micropipet moved upwards 5 l.un (as observed on the LCD display of the micromanipulator), and the z-axis limit set. The pipet is now positioned 30-32 l.un above the bottom of dish, so that the tip will clear the top of the neurons. Neurons are positioned under the tip of the mjection micropipet using the x-y movement of the microscope stage. The focus of the microscope is adjusted back and forth between the neuron and the pipet tip to assess the relative position The injection sequence is inmated with the foot swrtch when the neuron IS positioned with the pipet tip about 0.25 diameter from the edge of the neuron. A successful injection results in a drstmct blanching (i.e., a change m refractive index) of the cell interior and rapid increase in the diameter of the neuron (-10%). Injection can be confirmed by checking the cell for fluorescence of the coinjection marker. Sometimes repositioning the neuron so that the pipet tip is closer to the center of the cell (where it sticks up more) results m a successful Injection when the initial attempt is unsuccessful. Subsequent neurons in the dish are injected by movmg the stage to reposition the next neuron. Use a scanning pattern to systematically cover a given area of the dish. Approximately 50 neurons in a dish can be injected in 1O-l 5 mm. Neurons are returned to the incubator following mjectton. Although mjections take place under only pseudostenle conditions, microbial contaminatton has not been a problem in the short term (24-36 h). Following overnight incubation, prevtously injected neurons are identified from the fluorescence of the coinjectton marker. It is prudent to mmimize the exposure of the preparation to the Intense eptfluorescent illummation as products resulting from photobleaching might be toxic. Patch-clamp recordings are performed on the injected neurons 8-24 h postisolation. At this time, the neurons are stall relatively spherical and usually devoid of significant processes, thus facilitating whole-cell voltage-clamp recordmgs.

3.3. injection

of cDNA into the Nucleus of Neurons

1. Injection of cDNA into the nucleus of sympathetic neurons utihzes methodology common to that described above for cytoplasmic injections, so only major dtfferences in protocol will be described m detail. 2. DNA coding for the desired protem, subcloned mto an appropriate mammalian expression vector (see Note lo), is stored at -2O’C at a concentratton of 1 clg/& in TE buffer. 3. A reporter plasmid encoding the S65T mutant (8,9) of the Aequroia victoria green fluorescent protein (GFP; see ref. 10) is coinjected to facilitate identification of neurons receiving a nuclear injection (see Note 11). 4. 1 pL of plasmid DNA (supercoiled) encoding the target protein (1 c(g/l.tL stock) and the S65T GFP (1 ug/& stock) are mixed with 8 l.tL of TE buffer (pH 8.0). The solution is centrifuged as described above (see Section 3.2., step 5). 5. DNA solutions are kept at room temperature during the injectton session.

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6. Injection pressure (P2) usually ranges from 100 to 200 hPa, and injection duration from 0.2-0.3 s, depending on pipet size. 7. Injections are carried out as for cytoplasmic injections, with the following differences: The neuron is posrtioned so that the pipet tip is centered over the middle of the nucleus. Successful nuclear injections are difficult to confirm vrsually. The best indication seems to be a bright dot that forms in the center of the nucleus. If the nucleoli move laterally during the injection, this usually means the nuclear membrane was dented but not penetrated. Pronounced swelling of the cell almost always means a cytoplasmrc injection. Significant swelling of the nucleus is poorly tolerated and neurons experiencing such treatment often die Injection prpets tend to clog much more frequently when compared with cytopiasmtc RNA injecttons This tendency seems to result from both the properties of plasmld DNA (less soluble) and the consistency of the nucleus (very viscous). 8. Followmg overnight incubation, neurons that recerved successful nuclear injections are identified from the fluorescence of the heterologously expressed S65T GFP. The green fluorescence is usually easily detected under the conditions described above (i.e., Nikon B2A filter cube). If the fluorescence is dim, the culture media should be removed and replaced with a solution that does not contain phenol red, which mcreases the background fluorescence. 9. In our hands, nuclear injection of adult rat sympathetic neurons is much more difficult than cytoplasmic injection. We estrmate that perhaps 10% of attempted nuclear injections result in a satisfactory expression. The problem seems to arise from locating the nucleus in the z-axis. Thus, the large size of the neurons, an asset for cytoplasmic injections, is actually a hindrance for nuclear injections. It should be noted, however, that the same receptors (e.g., mGluR3) that did not produce functional expression following cRNA injection generated robust response following nuclear mjection of cDNA.

4. Notes 1. The enzyme concentrations must be adjusted empirically for each new lot of collagenase and trypsin. Thus, proteolytic enzymes should be purchased m large quantities (several grams) once suitable lots are identified For long-term storage (years), we ahquot the enzymes and store them at -80°C. The working aliquot of collagenase and trypsin IS stored at 4°C. Collagenase and trypsin are used at concentrations of OS-l.0 and 0 3-O 5 mg/mL, respectively The concentration of DNase is not critical. 2. We have used trypsm from Boehrmger Mannheim for many years and have yet to find a trypsin from another manufacturer that seems to work as well for this application. Unfortunately, Boehringer Mannheim no longer sells thus trypsm in the United States, though it is still available in Europe. 3. The anatomy of the rat autonomic nervous system, includmg supertor cervtcal ganglion, is illustrated by Gabella (11). 4. The vigorous shake method of neuron dissociation (see Section 3.1 2., step 4) was developed by the author and Dr. G. G. Schofield (12). We prefer this method

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to the more commonly used method mvolving trituration of the cell suspension with a fire-polished Pasteur plpet. If large clumps of tissue remain after shaking the flask, a higher concentration of enzymes is required. The concentration of trypsin seems to have the greatest influence on the quality of the preparation; thus, this should be increased first Conversely, the trypsin concentration should be decreased if the neurons perish during the first few hours of incubation, assume a shrunken appearance after dtssociation, or retain multiple long (several cell diameters) processes. Initially, it is probably easiest to use commercially manufactured ptpets (i.e , Femtotips) for injecting. Manufacturing your own pipets, however, is considerably less expensive (if you have access to a pipet puller) and allows for some customization of pipet geometry. Start by observing the overall geometry of a Femtotip under a microscope at low (60x) and high power (600x). Then try to reproduce the sharply tapering geometry with a ptpet puller. We pull injection micropipets from thin-walled (od, 1.2 mm; id, 0.9 mm; length, 100 mm) borosilicate capillary-filled microelectrode blanks (cat. no. KTW120F-4, World Precision Instruments, Sarasota, FL) using a Brown-Flamming type pipet puller (P-83, Sutter Instrument, Novato, CA). The pipet glass is cleaned and baked as previously described for hematocnt capillaries (see Section 2.2., item 4). A three-stage pull is used, with settings of heat, pull, velocity, and time: (a) 780,0,30,200, (b) 790,0,30,200; and (c) 800, 130, 18, 150. These settings vary considerably with puller (even of the same model), batch of glass, and filament type (we use a boxtype platmum filament), but may represent a reasonable starting point. Since the opening of the micropipet is too small to observe with a light microscope, the suttability of mtcropipets must be tested functionally (i.e., by mjectmg a few cells) Because culture dishes are not perfectly flat, the z-axis mjectton limit usually has to be adjusted if the dish is moved far from where the initial setting was determined. This can be a major irritation if the distance between individual neurons is great. The use of a cloning ring to confine the neurons to the center of the dish during plating muumtzes this problem. Cloggmg of the injection pipet is a frequent problem, especially with DNA solutions. Sometimes the offendmg particle can be ejected with a brief pulse of high pressure (PI button). A more desperate maneuver 1sto gently touch the tip of the pipet to the bottom of the culture dish using the step mode of the mtcromantpulator. This maneuver usually enlarges the opening of the tip, thus requiring a reduction of injection pressure. The trick works well for cytoplasmic injections, but not nuclear injections, We use mMessage mMachme m vitro transcription kits (Ambion, Austin, TX), per the manufacture’s instructtons, to make cRNA. Mammalian expression vectors containing a CMV promoter, such as pC1 @omega, Madison, WI), work well m SCG neurons High level expression seems to require injection of supercoiled plasmrd DNA into the nucleus. Linearized plasmid DNA or injection of plasmid DNA into the cytoplasm produces little or no expression. We use Qiagen (Chatsworth, CA) columns to purify plasmid DNA

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11. The S65T GFP (8) used in our studtes was subcloned into pCI. The translation initiation region was altered to conform to a Kozak consensus sequence (13) using the PCR. This resulted in a Ser-to-Gly mutation at the second amino acid. 12. The decision of whether to use cytoplasmic inJection of cRNA or nuclear injection of cDNA for expression in sympathetic neurons should be based on the following considerations. In general, cytoplasmic injections are much easier to perform and thus many more neurons are usually available for study. However, the manufacture of cRNA requires an additional step and the susceptibility of RNA to degradation requires more careful handling and storage. Conversely, nuclear injections are more difficult to perform and thus might provide a frustrating introduction to micromjection technique. However, we have been able to obtain functional expression of several constructs (receptors and G protein subunits) only from nuclear injection. Our impression is that expression levels resulting from nuclear injection of DNA are much higher than those obtained with cytoplasmic inJection of RNA. Consequently, nuclear inJection may be preferable when attempting to express dominant negative mutations. We speculate that the higher level of expression results from the stability of plasmid DNA in the nucleus, the strong constitutive promoter used (CMV), and the increased stability of nuclear-derived RNA because of posttranscriptional processing (e.g., addition of a poly-A tail). 13. Expression from either RNA or DNA can sometimes be enhanced by removing most of the untranslated regions from the clone (14). We find that start condons (AUG) in the 5’ untranslated region can be particularly troublesome (15).

References 1. Nakanishi, S. and Masu, M. (1994) Molecular diversity and functions of glutamate receptors. Annu. Rev. Biophys. Biomol. Struct. 23,3 19-348. 2. Pin, J.-P. and Duvoisin, R. (1995) The metabotropic glutamate receptors: structure and functions. Neuropharmacology 34, l-26. 3 Hofmann, F., Biel, M , and Flockerzi, V. (1994) Molecular basis for Ca2+ channel diversity. Annu. Rev Neuroscz. 17, 399-418. 4. Caulfield, M. P., Jones, S., Vallis, Y., Buckley, N. J., Kim, G.-D., Mulligan, G., and Brown, D. A. (1994) Muscarinic M-current inhibition via GaqllI and a-adrenoceptor inhibition of Ca2+ current via GaO in rat sympathetic neurones. J. Physzol. (Lond.) 477,4 15-422. 5. Law, S. F., Yasuda, K., Bell, G. I., and Reisine, T. (1993) Gla3 and G,, selectively associate with the cloned somatostatin subtype SSTR2. J. Blol Chem 268, lo,72 l-10,727. 6. Ikeda, S. R., Lovinger, D. M., McCool, B. A., and Lewis, D. L. (1995) Heterologous expression of metabotropic glutamate receptors m adult rat sympathetic neurons: subtype specific couplmg to ion channels. Neuron 14, 1029-1038. 7. Ikeda, S. R. (1996) Voltage-dependent modulation of N-type calcium channels by G protein &-subunits. Nature 380,255-258.

202

lkeda

8 Heim, R., Cubit& A. B , and Tsien, R. Y. (1995) Improved green fluorescence. Nature 373,663,664.

9 Cubitt, A. B., Heim, R , Adams, S R , Boyd, A. E., Gross, L. A., and Tsien, R. Y (1995) Understandmg, Improving and usmg green fluorescent proteins Trends Biochem

Scl 20,448-455.

10. Chalfle, M., Tu, Y., Eusknchen, G., Ward, W. W , and Pracher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Sczence 263,802-805. 11. Gabella, G. (1985) Autonomic nervous system, in The Rat Nervous System, vol 2, Hindbraln and Spinal Cord (Paxmos, G , ed.), Academic, New York, pp 325-353. 12. Ikeda, S. R., Schofield, G. G., and Weight, F. F. (1986) Na+ and Ca” currents of acutely isolated adult rat nodose ganglion cells. J Neurophysiol. 55,527-539. 13. Kozak, M. (1986) Pornt mutations define a sequence flankmg the AUG initiator codon that modulates translation by eukaryotic rtbosomes. Cell 44,283-292. 14 Kaufman, R. J. (1990) Vectors used for the expression in mammalian cells Methods Enzymol. 185,487-5 11. 15. Pantopoulos, K., Johansson, H. E , and Hentze, M .W (1994) The role of the 5’ untranslated region of eukaryotic messenger RNAs m translation and its investtgation using antisense technologres. Prog. Nut Acid Res Mol. Biol. 48,18 l-238.

15 Nuclear Application of Antisense Oligonucleotides by Microinjection and Ballistomagnetic Transfer to Identify G Protein Heterotrimers Activating Phospholipase C Frank Kalkbrenner, Edgar Dippel, Matthias Schroff, Burghardt Wittig, and Giinter Schultz 1. Introduction G proteins link heptahelical membrane receptors to their effector systems. The G proteins consist of three subunits, a, /3, and y, of which until now 23

(including splice variants), 6, and 11 different forms are known, respectively (for reviews, see refs. I and 2). By sequence homology of G protein a-subunits, they are divided into four subfamilies, G,, G,, G,, and Gr2. Agonist bindmg is assumed to induce a conformational change of the receptor, which causes exchange of GDP for GTP at the G protein a-subumt,

dissociation

of GaPy

from the activated receptor and dissociation of the Ga-GTP complex from the Gfiy dimer. Both activated Gc+GTP and free G& have the capability of interacting with different effecters, e.g., adenylyl cyclases, phospholipases Cj3 (PLCP), and ion channels. It is of growing interest to establish whether receptors may be able to select among individual a-, p-, and y-subunits for coupling to specific effector systems. Antisense technology allows selective inhibition of the expression of one particular protein involved in signal transduction and investigation of which cell function is altered (3,4). Using this technique, we determined the G proteins of a particular subunit composition that mediate hormone-induced inhibition of voltage-operated Ca2’ channels (5-9) or stimulation of PLCP, the latter measured as increase in the intracellular calcium concentration ([Ca2’]J (10). In the latter studies, we used as a model system rat basophiltc leukemia cells stably transfected with the ml muscarinic receptor (RBL-2H3-hml) (12). From

Methods I/J Molecular Bmlogy, vol 83 Receptor S/gna/ Transductm Ed&d by R A J Chalks Humana Press Inc , Totowa, NJ

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To answer the question of which G protein subunits are, specifically, involved in the coupling of the muscarmic MI and adenosine A3 receptor to PLCP, we inhibited the expressron of G protein subunits by mtranuclear microinjection of antisense oligonucleotides complementary to sequences of G protein subunit mRNAs into RBL-2H3-hml cells. As an index of PLCP activity, the increase in [Ca2+], was measured by single-cell imaging of the injected cells loaded with Fura-2. Our results indicate that the Mr receptor selectively couples to PLCP via a G protein complex composed of GaqlI t PI,4 ~4;the endogenously expressed A, receptor selectively couples via G protein heterotrimers composed of Ga13P2y2(ref. IO; Dippel et al., manuscript in preparation). By using the same methods i.e., microinjection of antisense ohgonucleotides in combrnation with single-cell fluorescence determination of the increase in [Ca2+],, we determined the subunit composition of the G proteins coupling ar-adrenoceptors to calcium increase in primary myocytes from rat portal vein as ad1 1 p1,3~213 (12). Table 1 summarizes the functional G protein heterotrimers determined by this method. Recently, we have developed a method to introduce antisense oligonucleotides simultaneously into a large number of cells, employing a method termed ballistomagnetic transfer, to investigate G protein-effector coupling when no method is available to assessactivity at the single cell level. Here we present a detailed description of the methods of nuclear microinjection and ballistomagnetic transfer of antisense oligonucleotides directed against G protein a-, j3-, and y-subunits, for measuring increases in [Ca2’], in single cells as an index of PLCP activity, and for establishing the extent of antisenseinduced G protein reduction by immunofluorescence staining of G protein subunits. To illustrate these methods, we will take as an example the identification of G protein heterotrimers that link M, and A3 receptors to PLCP in RBL-2H3hml cells. 2. Materials

2.7. Selection and Microinjection

of Oligonucleofides

1. The sequencesof oligonucleotidesusedin previous publicattons (5-10) andcorresponding target sequencesin mRNAs of G protein subunitsare summarizedin Degtiar et al. (13) or in the original publications.They were chosenby sequence comparison andmultiple alignment using MacMolly Tetra software (Soft Gene, Berlin). When the basesequencesof rat mRNAs were not known for the G protein subumtsunder investigatron, we used the statisticalapproachof preferred codon usagein the rat to obtain the most likely sequences(seeNote 1). 2. For injection into different cell lines, we previously used antisenseohgonucleotideswith phosphodiesterboundsbetweenthe nucleotidesor partially protected oligonucleotides(i.e., in the last two nucleotidesat both the 5’ and 3’ ends,one of

205

An tisense Oligonucleo tides Table 1 Summary Coupling

of G Protein Heterotrimers Hormone Receptors to Phospholipase

Receptor ml Muscarinic A3 Adenosine a 1 Adrenergtc

C-p

G protein heterotrimer ~qmP1/4Y4 %3P2y2 %/I

1P 1/3y2/3

Cell line RBL-2H3-hml RBL-2H3-hm 1 Rat portal vein myocytes

the nonbridgmg oxygens of the phosphate group was replaced by sulfur [phosphorothioates] to protect the ohgonucleotides from degradation by exonucleases). For the injection into the nuclei of RBL-2H3-hml cells and rat portal vein myocytes, we used completely phosphorothioate-protected oligonucleotides (see Note 2). Oligonucleotides were synthesized by a DNA synthesizer (Milligen model 8600, Millipore, Eschborn); for synthesis of phosphorothioate oligonucleotides, the method described by Iyer et al. (14) was used. 3. We used commercial pipets (Femtotips, Eppendorf, Hamburg) or pipets pulled from borosilicate glass tubes with filament (od, 1.12 mm; id, 0.96 mm; Hilgenberg, Malsfeld, FRG). The outlet tip diameter was approx 0.5 pm for the Eppendorf pipets and 0.5-l .O pm for the Hilgenberg pipets.

2.2. Measurement

of Cytosok

#+

1 Fura- loading buffer: 138 mMNaCl,6 mA4KC1,O. 1 mMCaCla, 1 mMMgS04, 1 m&f Na2HP04, 5 mM NaHC03, 5.5 mM glucose, 20 mA4 HEPESNaOH, pH 7.4, containing 0.1% (w/v) BSA and 2 @fmra-2/acetoxymethylester (MoBiTec, Gdttingen, FRG). 2. Wash buffer: 138 mM NaCl, 6 mM KCl, 1 mA4 MgC&, 1 mM CaCl,, 5 5 mA4 glucose, 20 mMHEPES/NaOH, pH 7.4. 3. Fura- calibration solutions: 10 @4 ionomycm and 2 mA4 CaC12; and 10 pA4 ronomycin and 10 mA4 EGTA. Each solution is made up in wash buffer.

3. Methods 3.1. Microinjection

and Calcium Measurements

3.1-I. Microinjection of Oligonucleotides 1. One day prior to injection, RBL-2H3-hml cells were seeded at a density of about 1 x lo3 cells per mm2 on cover slips imprinted with squares for localization of injected cells. 2. Injections of oligonucleotides were performed either by an automated (AIS, Zeiss, Oberkochen) or a manual injection system (Eppendorf). 3. The injection solution routinely contained 10 pA4 oligonucleotides m water; other concentrations (5 or 20 pA4) used for some experiments did not influence the results.

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4. The increase in nuclear and entire cell volumes were used asa visual control for successful injection (presumably 10-20 fL were injected). To measure the microinjection efficiency, cells were injected with a 10 @4 solution of fluorescein isothiocyanate (FITC)-marked oligonucleotides The fluorescence signal remained for 48 h in the nuclei of about 90% of the inJected cells, although its intensity (reflecting the amount of injected oligonucleotldes) varied from cell to cell. 5. Following injection, RBL-2H3-hml cells were usually cultured for 46-76 h before Fura- calcium measurements were performed. After injection, the cells were cultured in culture medium containing only 2% fetal calf serum (FCS) for 36 h, in order to reduce proliferation of cells (see Note 3). Twelve hours before fluorometric measurements, cells were substituted with regular culture medium containing 18% FCS.

3.1.2. Measurement of Cytosolic C&+ Since no method is available to determine the PLCP activity in single cells, we measured the hormone-induced increase m cytoplasmic Ca2” as an index of PLCP activity. 1. RBL-2H3hml cells were loaded with 2 @f fura-2/acetoxymethylester (MoBiTec, Gbttingen) for 60 min at 37°C in loading buffer. Cells were then washed twice with 2 mL of the wash buffer. 2. For determination of cytoplasmic Ca2+, cells were overlayered with 300 ).tL of the wash buffer, supplemented with 1.1 mM EGTA. Determinations of Ca2+ m single cells were performed at 37’C, using a digital imaging system (T.I.L.L. Photonics, Mhnchen, FRG). Cells were visualized using an inverted microscope (Zeiss Axiovert 100) and a Neofluor 16x oil immersion lens (Zeiss). 3. Fura- fluorescence was excited alternately at 340 and 380 nm with illummation

provided by a 100 W xenon lamp. Cellular fluorescencewas filtered through a 5 10-m band pass filter. 4. For each single cell measured, F,,, and F,,, were determined by subsequent

addition of buffer containing ionomycin (10 @4) and CaCl, (2 mM) and buffer containing ionomycin (10 @4)and EGTA (10 mM). Imageswere dtgitalized and analyzed by the software Fucal5.14 (T.I.L.L. Photomcs) Ratio images were generated at 0.5- to 1-s intervals. For background compensation, illumination of an area containing no cells was subtracted. For each cell, [Ca2+], was averaged from pixels within manually outlined areas.

3.2. Control of Specificity of the Injected Ant/sense OIigonuckotides 3.2.1. lmmunocytochemistry In order to be sure that injection of antisense oligonucleotides directed against specific G protein subunits led to suppression of the expression of the

Antisense Oligonucleotides A

non-injected

207

anti-aqlll

Fig. 1. Inhibition of Ga,,i , protein expression in RBL-2H3-hml cells injected with anti-ag+, i antisense oligonucleotides. The panel shows noninjected cells (left) and cells injected with anti-ap+, I antisense oligonucleotides (right) on the same cover slip. All cells were stained 48 h after injection with rabbit anti-a,,, t antiserum (AS 370) (1: 100) specific for Go,,, ,, and visualized by staining with FITC-conjugated goat anti-rabbit IgG (1: 1000). Data were taken from ref. 10.

subunits, we determined the expression of G proteins in injected cells by immunofluorescence experiments. The following protocol was used to stain injected cells with polyclonal antibodies directed against peptides derived from GCQ 1 or individual Ga;-subunits:

respective

1. Cells were washed with phosphate-buffered saline (PBS, pH 7.4), fixed with ethanol/acetic acid (10: 1, v/v) for 30 min at room temperature, washed again with PBS, and permeabilized for 60 min with 0.1% saponin in PBS containing 3% FCS 2. Cells were washed (3 x 10 min) with this buffer containing 5% FCS and incubated with the same buffer containing 3% FCS with, e.g., the polyclonal anti-G,,r , rabbit antibody (AS 370) at 1: 100 dilution overnight at 4°C. The cells were then washed in PBS (4 x 10 min) and incubated with goat antirabbit IgG conjugated to fluorescein isothiocyanate (diluted 1: 1000 or 1:2000) in PBS containing 5% FCS and 0.1% saponin for 18 h at 4’C. The cells were then washed (4 x 10 min) in PBS and mounted in Moviol (Hoechst, Frankfurt, FRG). 3. Images of the stained cells were obtained by a video imaging system, using monochromatic light at 495 nm and an exposure time of 1 s. To prevent bleaching, cells were not exposed to light at 495 nm before pictures were taken. 4. The average of pixels outlined from the immunofluorescence of the cell membrane were corrected for background illumination and calculated for each cell by constant acquisition parameters. The digitalized fluorescence images were visualized by the software NIH Image (Wayne Rasband, NIH, Washington, DC). 5. In cells injected with a mixture of anti-ag and anti-a,, antisense oligonucleotides, the amounts of the corresponding immunochemically detected proteins were reduced by 85% after 2 d (Fig. 1). The expression of Gas and Go.,, completely recovered after 4 d (see refs. 4 and 10).

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3.2.2. Specificity of the Antisense Oligonucleotides In order to have an internal control for the specificity nucleotides in each experiment:

of the antisense olrgo-

1. We compare injected cells located within a marked area on the cover slip to noninjected cells located outside of the marked area (see Note 4). 2 Demonstrate the specificity of the antisense oligonucleotide (see Note 5)

Both types of experiment, comparing antisense-injected cells to each other and comparing anttsense-injected cells to noninjected cells, are, in our opinton, a prerequisite for the validation of the specificity of the antisense oligonucleotides used. In addition, both kinds of control experiments should be performed on cells located on the same cover shp at the same time. 3.3. Ballistomagnetic

Transfer of Antisense

Oligonucleotides

The ballistomagnetic vector system can transfer nucleic acids and other biomolecules into the nuclei of populations of cells (5 x 107-5 x lo*) efficiently, and transfected cells can be obtained at >90% purity in less than 60 min. The system is of great importance for G protem-coupled effector systems for which no appropriate method of measurement in single cells is available. The ballistomagnetic vector system employs a two-step procedure: First, the actual transfer of nucleic acids, together with supraparamagnetic particles into the nuclei of many cells; second, the magnetic separation of actually transfected cells from nontransfected cells (15,16). Figure 2 shows the principle of this method schematically. 3.3. I. Ballis tic Transfer 1. A suspension of colloidal gold (0.5 mg, 1 6 mm, Bio-Rad, Mumch, Germany) 1s pipeted onto each of seven particle carrier membranes (purchased as macrocamer from Bio-Rad) and allowed to sediment. 2. After removal of the supernatant, the gold particles are resuspended in a mixture of three parts of an aqueous solution of DNA and two parts of a suspension of colloidal supraparamagnetic particles (65 nm diameter, Miltenyi GmbH [Bergisch Gladbach], used as purchased). The suspension was allowed to sediment, the supernatant was removed, and the particle carrier membranes were kept at room temperature until residual liquid had completely evaporated. 3. The accelerating system for ballistic transfer is based on the blohstic PDS- 10001 He apparatus (1550 psi rupture disk, 500 mm Hg of vacuum, Bio-Rad). The btohstic unit was modified by a pressure outlet manifold, a multipartrcle carrier assembly, and an adjustable bearmg to carry a lo-cm Petri dish 4 Ballistomagnetic transfer of biomolecules into 107-3 x 10’ cells seeded onto lo-cm Petri dishesis achieved by simultaneousdelrvery of particles from seven particle carrier membranes that are arranged in a way to cover the entrre area of

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Antisense Oligonucleotides helium

para,magnetic

beads covered with dextran

antlsense

ollgonucleotides

gold particle

sorting by column and magnet

IY c3 Ga non-magnetic

fraction

magnetic fraction

Fig. 2. Schematic illustration of the ballistomagnetic vector system for the transfer of antisense oligonucleotides into RBL-2H3-hml cells. the Petri dish evenly. Cell culture medium is removed from the Petri dish immediately prior to operating the ballistomagnetic vector system.

3.3.2. Magnetic Separation 1. A high-gradient magnetic separation column (capacity 3 x lo7 cells, type AS, Miltenyi GmbH) is prepared according to the supplier’s protocol. Following

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2.

3.

4

5.

ballistomagnetic transfer, cells are immediately resuspended m 2 mL PBS, supplemented with 0.25% of bovine serum albumin, and transferred onto the column. An aliquot of the cell suspension IS kept for the reference (unsorted fraction) The cell suspension is passed through the high-gradient magnetic separation column, followed by a washing step with 3 mL PBS. The effluent is collected (negative or nonmagnetic fraction). Following removal of the column from the magnetic separator, the retained cells are flushed back to the top of the column. The column is put back into the separator, washed with 3 mL PBS, and the effluent collected (wash fraction). Finally, the column is again removed from the separator and eluted with 5 mL PBS (magnetic fraction). The fractions collected are further processed according to experimental conditions required for the subsequent assay (see Notes 6 and 7). Examples of data obtained using the ballistomagnetic vector system to transfer of FITC-marked ohgonucleotldes (see Note 6), and transfer Gas+, , antisense ohgonucleotides (see Note 7) into RBL-2H3-hml cells are described.

3.4. Conclusions and ballistomagnetic transfer are the only methods that result in an intracellular excessof antisense oligonucleotides and thus cause a significant reduction of G protein function. In our recent studies, we have demonstrated the extent and specific@ of In our experience,

suppression

microinjection

of G protein

subunit expression by antisense oligonucleotides

if

directly transferred into the nuclei of RBL-2H3-hml cells. In particular, suppression of one subtype of G protein subunit can selectively block signal transduction

from one receptor, while signaling

from another receptor through

a

closely related pathway to the same effector system remains unaffected. Using sequential specificity

applications of two different of the injected oligonucleotides

hormones, we are able to prove the for each cell under investigation. In

addition, the normal response of a cell to one of the hormones used demonstrates the viability

of the cell. It is our opinion

that antisense experiments

should generally include three kinds of control experiments: comparison of injected cells with noninjected, or with control cells injected with other oligonucleotides on the same cover slip; comparison of two related pathways in the same cell, both allowing for internal controls, as described in Notes 4 and 5; and the ability

of antisense oligonucleotides

to suppress the expression of the

targeted protein should also be proven. Thus, microinjection or ballistomagnetic transfer of anttsense oligonucleotides in combination with fluorometric detection of hormone-induced changes in [Ca2+], provides a powerful and important tool for the identification of signal transduction cascades.Targeting the individual G protein sub-

Antisense Oligonucleotides

211

units by the anttsense knock-out has led to the identification of strict requirements for receptor-G protein-effector coupling within the membrane environment (4).

4. Notes 1. When the base sequences of rat mRNAs were not known for the G protem subunits, we used the statistical approach of preferred codon usage m the rat to obtain the most likely sequences. First, we translated the respective mRNA base sequence as sequenced from one species into the corresponding amino acid sequence. This step does not create uncertainty, since the genetic code is definite in translation. Second, we translated the unambiguous amino acid sequence back mto mRNA base sequence. Here, we used the evolutionary preferred codon usage of the rat. By using the targeted species codon usage, the degeneracy of the genetic code is reduced considerably. The obtained nucleotide sequence is unambiguous in about 95% of the bases, compared to >60% if the universal codon usage is used. The resulting nucleotide sequence was used to run an anttsense oligonucleotide search program under highly stringent conditions, which allowed a chorce of either unambiguous antisense sequences, or sequences with wobbling in as few positions as possible. 2. With these completely phosphorothioate-protected oligonucleotides, we observed nonspecific effects only at concentrations above 50 @4. 3. Low serum levels m the culture medium were necessary to keep the cells as separated single cells during incubation after injection, to avoid development of a monolayer of cells. 4. In order to have an Internal control for the spectflcity of the antisense oligonucleotides in each experiment, we compare injected cells located wtthm a marked area on the cover slip to noninjected cells located outside of the marked area. This guarantees that injected cells are always compared to control cells that are otherwise treated in the same way, e.g., incubation, microinjection, loading with Fura-2, and measuring for increase in [Ca2’],, all at the same time and under identical experimental conditions. Figure 3 shows original traces of cells injected with antisense oligonucleotides directed against the y2- (anti-y2) or y4-subunit (anti-y& Cells were either stimulated with the adenosine receptor agonist NECA or with the muscarmic receptor agonist carbachol. Cells inJected with anti-y2 antisense oligonucleotides showed a significant reduction of the increase in [Ca2’], induced by NECA compared to noninjected cells; in cells injected with anti-y4 antisense oligonucleotides no difference in the NECA-induced increase in [Ca2’], compared to noninjected control cells was found (Fig. 3A). In contrast, cells injected with anti-y4 antisense oligonucleotides displayed a significantly lower increase in [Ca2+], induced by carbachol compared to control cells; cells injected with anti-y2 antisense oligonucleotides showed no difference in the carbachol-induced increase in [Ca2’], compared to noninjected control cells (Frg. 3B). Table 1 shows the subunit composition of the G protems involved in coupling of both receptors to PLCP.

Kalkbrenner et al.

212 500 450 400 350 300 250 200 150 100 50

-I 600 8 L 0

10

20

30

40

50

0 60

70

6

400

300

300

2

200

200

F0

loo

100

0

50

100

30

40

50

60

time [s]

500

400

0

20

anti-y4

500

.3 52 8

=

IO

BOO 1

anti-y2

E

0

-c----l

o150

0

50

100

150

time [s]

Fig. 3. (A) NECA-induced increase in [Ca*‘], in RBL-2H3-hml cells injected with anti-y? or anti-y, antisense ohgonucleotides. (B) Carbachol-induced Increase in [Ca*+], in RBL-2H3-hml cells injected with anti-y2 or anti-y4 antisense oligonucleotides. The NECA and carbachol concentrations used were 10 @4 and 1 mh4, respectively. The traces show average trme-courses of about 15 injected and uninjected cells, each measured on the same cover slip. Data were parttally taken from ref. 10. 5. A second, important internal control of the specificity of the antisense oligonucleotides IS to show that injection of one partrcular antisense oligonucleotide (i.e., suppressron of one subtype of G protem subunit) selectively blocks signal transduction from one receptor, while signaling from another receptor, through a closely related pathway to the same effector system (i.e., receptor-mediated increase in [Ca*‘],), remains unaffected. For this purpose, we use sequential applications of two different hormones on cells inJected with antisense oligonucleotides blocking the signal transduction pathway induced by one hormone, but without effect on the signal transduction pathway induced by a second hormone. For instance, we have compared the MI-receptor-induced increase in [Ca*“], with that induced via adenosine A3 receptors in RBL-2H3hml cells. The adenosine receptor-agonist NECA (10 @I) induces a PTX-sensitive increase in

213

Antisense Oligonucleotides Carbachol 500 -

NECA

I

O-l 0

2

4

6

time [min] Fig. 4. NECA- and carbachol-induced increases in [Ca2+], in RBL-2H3-hml cells injected with anti-ag+, , or antl-a,common antisense oligonucleotides. Each trace repre-

sentsthe time-course from one cell either injected with anti-aq+l, or antiqcommon. The arrows indicate the application of NECA (10 @4) and carbachol(1 mM).

[Ca2’], in RBL-2H3-hml cells. Since Gi2 and G,3 are the only PTX-sensitive G proteins expressed in this cell line, we injected cells with antisense oligonucleotides complementary to a common sequence of all three mRNAs encoding Ga, isoforms (anti-y,common). On the same cover slip, we injected cells located in another marked area with a mixture of two antisense oligonucleotides directed against GCL~and Gal1 (anti-olq+, J. Figure 4 showsthe results for two cells, one injected with anti-a,common antisense oligonucleotides and the other one injected with anti-o14+, , antisense oligonucleotides. After application of NECA, the anti-ol,common-injected cell showed a decreased peak of [Ca2+], compared to the anti-aqtl 1-injected cell; vice versa, after application of carbachol, the antiuqtl 1 antisense oligonucleotides-injected cell showed a reduced increase m [Ca2+]i compared to the anti-a,common-injected cell (Fig. 4). 6. The ballistomagnetic vector system has been used to transfer FITC-marked oligonucleotides into RBL-2H3-hml cells. In control experiments, we transfected RBL-2H3-hml cells with fluorescein isothlocyanate (FITC)-labeled oligonucleotides and separated them as described in Section 3.3.2. Figure 5 shows a fluorescence-activated cell sorter (FACSscan) analysis comparing the cells of the nonmagnetic fraction (Fig. 5A) to cells of the magnetic fraction (Fig. 5B). Out of the cells of the nonmagnetic fraction, only 15% showed

fluorescence, e.g., were transfected with FITC-labeled remaining 85% showed no fluorescence. Cells from the more than 90% showed fluorescence and only 5-10% cence. These results indicate that, by using the method

oligonucleotides; the magnetic fraction by showed no fluoresof ballistomagnetic

Kalkbrenner et al.

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fluorescence

intensity [arbitrary units]

Fig. 5. FACSscan analysis of RBL-2H3-hml cells transfected with FITC-labeled oligonucleotides using the ballistomagnetic vector system (A) Cells of the nonmagnetic fraction (those not penetrated by the gold/magnetic particles). (B) Cells of the magnetic fraction (those penetrated by gold/magnetic particles). For details, see Section 3.3.

transfer, up to 90% of the cells can be successfully transfected with antisense oligonucleotides. 7 We have also transfected RBL-2H3-hml cells with a mixture of antisense ohgonucleotides directed against Gag- and Go, l-subunlts of G proteins (anti-o,,, ,) using the balhstomagnetic transfer method. Followmg separation of the magnetic and the nonmagnetic cell fractions, cells were cultured for 2 d on glass cover slips. The effects of applying either carbachol or NECA to coverslips are illustrated in Fig. 6, which shows superimposed traces of mean time-courses of 15-20 cells of each measurement Cells of the magnetic fraction transfected by ballistic transfer of antisense ohgonucleotides directed to the mRNAs encoding Go, and Gclr r showed much reduced carbachol-induced [Ca2’], peaks compared to cells of the nonmagnetic fraction. The NECA-induced signaling pathway was not affected. Cells of the nonmagnetic fraction, e.g., untransfected cells, responded equally to each agent

Acknowledgments We thank Rita Hauboldt for excellent technical assistance, Katrin Btittner for synthesis of oligonucleotides, Dr. Karsten Spicher for providing antisera (especially for the unpublished AS 370), and Dr. Penelope Jones for providing the RBL-2H3-hml cells. E. D. was a recipient of a Fellowship from the Deutsche Forschungsgemeinschaft. This work was supported by grants of the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.

215

Antisense Oligonucleotides 3OO

carbachol

NECA

T

non-magnetic

1

fraction

05 0

20

40

60

80

100

120

time [s] Fig. 6. Carbachol- and NECA-induced mcrease in [Ca2’], in RBL-2H3-hml cells transfected with ant++, 1 antrsense ohgonucleotides using the balhstomagnetrc vector system. The NECA and carbachol concentrations were 10 fl and 1 miI4, respecttvely. The dashed line represents cells of the magnetic fraction, the solid lme represents cells of the nonmagnetic fraction. The arrows indicate the time of application of the indicated agonist Each trace represents the average time-course of 15-20 cells,

References 1. Neer, E. J. (1995) Heterotrtmeric

G proteins: organizer of transmembrane signals

Cell 80,24%257.

2. Gudermann, T., Kalkbrenner, F., and Schultz, G. (1996) Diversity and selectivity of receptor-G protein interaction. Annu Rev Pharmacol. Tox~col 36, 429-459.

3. Albert, P. R and Morris, S. J. (1994) Antisense knockouts: molecular scalpels for the dissection of stgnal transduction. TrendsPharmacol. SCL15,250-254. 4. Kalkbrenner, F., Dippel, E., Wtttig, B., and Schultz, G. (1996) Specificity of interaction between receptor and G protein: use of antrsense techniques to relate G-protein subunits to function. Brochlm. Biophys Acta 1314, 125-139. 5. Kleuss, C., Hescheler, J., Ewel, C., Rosenthal, W., Schultz, G., and Wtttig, B. (199 1) Assignment of G protein subtypes to specific receptors inducing inhibition of calcium currents. Nature 353,43-48. 6. Kleuss, C., Scheriibel, H., Hescheler, J., Schultz, G., and Wittig, B. (1992) Drfferent b-subunits determine G protein interaction with transmembrane receptors. Nature 358,424-426.

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7 Kleuss, C., Scheriibel, H., Hescheler, J., Schultz, G., and Wittig, B. (1993) Selectivity in signal transduction determined by y subunits of heterotrimeric G proteins. Science 258,832-834. 8, Gollasch, M., Kleuss, C., Hescheler, J., Wittig, B., and Schultz, G. (1993) G,, and protein kinase C are required for thyrotropin-releasing hormone-induced stimulation of voltage-dependent Ca2+ channels in rat pituitary GH, cells. Proc. Natl. Acad. SCI. USA 90,6265-6269 9. Kalkbrenner, F., Degtiar, V. E., Schenker, M., Brendel, S., Zobel, A., Hescheler, J., Wittig, B., and Schultz, G. (1995) Subunit composition of G, proteins functionally coupling galanin receptors to voltage-gated calcium channels. EMBO J. 14,4728-4737.

10. Dippel, E., Kalkbrenner, F., Wittig, B., and Schultz, G. (1996) The muscarinic ml receptor couples to specific G protein heterotrimers to increase the cytosolic calcium concentration. Proc. Natl. Acad. Sci. USA 93, 139 1-1396. 11. Jones, S. V., Choi, 0. H., and Beaven, M. A. (1991) Carbachol induces secretion in a mast cell line (RBL-2H3) transfected with the ml muscarmic receptor gene. FEBS Lett, 289,47-50. 12. Macrez-Lepretre, N., Kalkbrenner, F., Schultz, G., and Mironneau, J. Distinct functions of G, and G, 1 proteins m coupling a,,-adrenoceptors to Ca2+ release and Ca2+ entry in rat portal vein myocytes. J. Biol. Chem., in press. 13. Degtiar, V. E., Wittig, B., Schultz, G., and Kalkbrenner, F. (1996) MicromJection of antisense ohgonucleotides and electrophysiological recording of whole cell currents as tools to identify specific G-protem subtypes coupling hormone receptors to voltage-gated calcium channels, in Transmembrane Signalling (Bar-Sagi, D., ed.), Methods in Molecular Biology, in press. 14. Iyer, R. P., Egan, W., Regan, J. B., and Beaucage, S. L (1990) 3H-1,2Benzodithiole-3-one 1,1-dioxide as an improved sulfurizing reagent in the solidphase synthesis of oligodeoxyribonucleoside phosphorothioates. J Am. Chem. Sk. 112, 1253,1254. 15. Sanford, J. C., Klein, T. M., Wolf, E. D., and Allen, N. (1987) Delivery of substances into cells and tissues using particle bombardment process. Particulate Sci. Technol. 5,27-37. 16. Miltenyi, S., Mtiller, W., Weichel, W., and Radbruch, A. (1990) High gradient magnetic cell separation with MACS. Cytometry 11,231-238.

Use of Antisense-Generating Plasmids to Probe the Function of Signal Transduction Proteins in Primary Neurons Fe C. Abogadie, Yvonne Vallis, Noel J. Buckley, and Malcolm P. Caulfield 1. Introduction Investigation of the function of intracellularly located protein components of receptor-effector transduction pathways has been hampered not only by their inaccessibility, but also by the lack of specific tools. The problem is particularly acute when the effect of interest must be measured at the single cell level, for example, receptor modulation of ion channels. Assignment of different G protein subunits to roles in ion channel modulation by receptors progressed greatly with the availability of antibodies raised against peptide sequences corresponding to the carboxyl-terminus of Go-subunits. This is the part of the a-subunit molecule thought to interact with effector, so these antibodies were predicted to indirectly occlude receptor-effector interaction. Indeed, we found that injection of an antibody against Go, into sympathetic neurons inhibited az-adrenoceptor effects on Ca2+current, and antibody against Ga,,r r inhibited muscarinic and bradykinin receptor effects on the M-type K+ current (1,2). However, this approach is seriously limited by the close sequence homologies between members of G protein a-subunit subfamilies (3), which results in the antibodies being unable to discriminate between members in many cases. Antisense oligonucleotides directed against parts of G protein sequences seemed to offer a specific solution. Specificity could seemingly be increased by using sequences directed against untranslated regions of G protein genes, in which interfamily homology is much less (4; see Note 1). Evidence for cellular From* Methods m Molecular Srology, vol 83 Receptor Signal Transductron Edlted by R A J Chalks Humana Press Inc , Totowa, NJ

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uptake of antisense oligonucleotides (5), and the use of chemical modifications to reduce intracellular or extracellular degradation (6), further enhanced the attractiveness of these tools. Intranuclear microinjection into pituitary-derived GH3 cells of various antisense oligonucleotides showed selective coupling of muscarinic and somatostatin receptors to inhibit calcium channel opening via Gael and Go+ respectively (4), at least asmeasured by functional, rather than protein changes. The notes for these chapters suggest that the literature rarely includes descriptions of “problems that can be encountered.. . and how they are identified and overcome. . .,” and that should be the purpose of these essays.In this chapter, we describe the productton of antisense RNA-generating plasmids, which we have now successfully used to investigate the role of various G protein a-subunits m coupling receptors to ion channels in neurons. The obvious question is why we used this approach, rather than the seemingly simpler antisense oligonucleotide strategy. The answer is that we have worked extensively with oligonucleotides, and found them wanting (that is not to say that critical and careful use of antisense oligonucleotides cannot generate useful data 161). We carried out a comprehensive and detailed functional study of muscarmic receptor modulation of M-type K’ current in sympathetic neurons, using anti-G protein sequences that had been effective m another system (71, and found highly significant effects of one antisense sequence at particular time-points. Attempts to duplicate these findings with other sequencesdirected against the same G protein gene failed, and as we did further experiments, the efficacy of the original effective sequence was found to be variable and unpredictable (Caulfield, Buckley, and Jones, unpublished). This has also been the experience of others (6). We felt that the use of plasmids to make antisense sequences would overcome at least some of the criticisms of antisense oligonucleotides, namely the uncertainties about duration of action and attainment of effective intracellular concentration, since the production of antisense sequence would be under the control of a strong viral promoter. Given that many of the original claims for oligonucleotide efficacy and selectivity were based solely on functional measures, rather than measurement of specific protein reduction (4), we also determined to combine demonstration of antisense plasmid efficacy, at both the protein and functional level, in single neurons. Clearly, a hurdle that must be overcome with antisense plasmids is that of delivering the construct mtracellularly, and this we have achieved with intranuclear microinjection. In this chapter, we provide details of how to go about generating antisense plasmids, with some notes on how we did our inJections in sympathetic neurons, and located our injected neurons for subsequent functional experiments. We also describe a general protocol for immunostaining of proteins against

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which antisense is targeted. We wanted to design plasmids that would produce antisense RNA against specific regions of target genes, but the sequences of the genes we were interested in (i.e., rat G proteins of the Go, and GoI2 families) are not known, so we had to clone these de y1ovo.For the initial PCRcloning, we used primers based on mouse sequences and did the PCR under low stringency conditions. The PCR products were directly cloned mto the TA cloning vector pCRI1 (Invitrogen). Because we wanted to express these constructs in mammalian neurons, we then subcloned these sequences into the mammalian expression vector pBK-CMV (Stratagene), which has the CMV viral promoter. More recently, the availability of the vector pCR3 (Invitrogen), which allows for direct cloning and eukaryotic expression, has saved us one subcloning step. 2. Materials 1. Tri Reagent-RNA/DNA/Protein isolation reagent (Molecular Research Centre Oxford, Horton-cum-Studley, Oxford, UK). 2. Enzymes, corresponding buffers, and oligo-dT,s primer (Promega, Southampton, UK). 3. Amersham DNA ligation system (Amersham, Little Chalfont, Buckinghamshire, UK) 4. TA cloning kit (Invitrogen, NV Leek, The Netherlands). 5. Electroporation cuvets (NBL Gene Sciences, Cramlmgton, Northumberland, UK). 6. DNase buffer (10X): 400 mM Tris-HCl, pH 7.9, 100 mMNaC1, 60 mM MgC12. 7. RT buffer (5X): 250 mMTris-HCl, pH 8.3,375 mA4KC1, 15 mA4MgCl,, 50 mA4 dithiothreitol. 8. Qiagen plasmid kit (Qiagen, Hilden, Germany) 9. Borosilicate electrode glass GC 150TF 10 (Clark Electromedical, Pangbourne, Berks, UK). 10. Ca*+-free extracellular solution, containing 0.5 fl tetrodotoxin (e.g , modified Krebs’ solution [Z]). 11. Fluorescein-isothiocyanate (FITC)-labeled dextran 70-kDa, lysine-fixable (Molecular Probes, Eugene, OR). Make 10% stock in water (this can be stored in a refrigerator). Note that the FITC-dextran solid is extremely hygroscoptc, and care must be taken to store it properly desiccated in the freezer. 12. High-vacuum grease (e g., Edwards high vacuum, also available from Sigma). 13. Appropriate specific prtmary antibodies against the protein, raised in species A. 14. Biotmylated secondary antibodies raised in species B against species A IgG. 15. ABC complex (Dako A/S, Denmark). 16. Serum from species B. 17. BCIP/NBT (Dako A/S) 18. Vectashield fluorescence mounting medium (Vector Laboratories, Peterborough, UK). 19. Protein or immunogenic peptide against which the primary antibody was raised. 20. Films: Kodak Ektachrome 320T-Tungsten balanced, and Ilford HPS Plus 400 (Ilford, Mobberley, Cheshire, UK).

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3. Methods 3. I. Preparation of Tissue RNA Extract RNA from rat cortex (see Note 2) using standard acid phenol guanidinium thiocyanate protocols for RNA preparations (e.g., Tri Reagent protocol, Molecular Research Centre Oxford). 3.2. DNase Treatment 1. Precipttate 10 pg RNA and dtssolve in 10 pL water. 2. Add: 5 uL 10X DNase buffer @omega), 29.5 pL water, 0 5 pL RNA& @omega, 33 U/pL), 5 pL RQl DNase (Promega, lU/pL), and incubate at 37°C for 15 mm. 3. Make up volume to 200 pL by adding 150 pL TE, pH 7.4. 4. Phenol/CHCls extract once, followed by CHC13 extraction 5. Precipitate RNA with 0.1 vol2A4 NaCl and 2.5 vol EtOH 6. Dry pellet and redissolve tn 5 pL water.

3.3. Reverse

Transcription

1. Assemble together: 2 pL DNased RNA (~2 pg [see Note 31); 0.5 pL oligo-dT (Promega, 0.5 pg/pL); and 7 5 pL water. 2. Denature at 65°C for 5 min, then quench on ice for 1 min. 3. Add 5 clr, 5 mMdNTPs, 4 pL 5X RT buffer (Promega), 0.5 pL RNAsin (Promega, 33 U/pL), and 0.5 JJL M-MLV RT (Promega, 200 U/pL) or AMV RT. 4. Incubate at 37°C for 1 h. 5. Heat kill enzyme at 80°C for 10 mm. Store cDNA at -20°C untrl ready for PCR amplification.

3.4. PCR Amplification 1. Add sequentially* 37.6 pL water, 5 ~.IL 10X PCR buffer wtth 15 mA4 MgC1, (Promega, to give a final concentration of 50 mM KCl, 10 mil4 Tris-HCl, 0.1% Triton X- 100, 1 5 mM MgCl*, pH 9 0), 2 pL 5 mM dNTPs, 2 pL 10 w forward primer, 2 pL 10 uA4 reverse prtmer, 1 pL RT reaction (5%), and 0.4 pL taq (Promega, 5 U/pL). Mix with pipet and cover with 50 pL light mineral oil. 2. Run PCR reaction. Try 95°C for 5 min, 63°C for 30 s, and 72°C for 1 min; followed by 28 cycles at 95°C for 30 s, 63°C for 30 s, and 72°C for 1 min; followed by 1 cycle at 95°C for 30 s, 63°C for 30 s, and 72’C for 7 min. These conditions are opttmized for 24-mer primers of 45-60% GC content (see Note 4). 3. Run 10 pL on a 1.5% agarose gel with size markers

3.5. TA Cloning The ligation procedure is adopted from the ligation protocol in Amersham’s DNA ligation system and Invitrogen’s TA cloning system (see Notes 5 and 6). 1. Assemble 1 JJL PCR product; 2 pL TA3 pCR3 vector (Invitrogen, 30 ng/pL); 4 pL 100 mA4 Tris-HCl, 5 mA4 MgCL, pH 7.6; 28 pL solution A (Amersham DNA ligation system); 7 pL solution B (Amersham DNA ligation system).

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2. Vortex. Incubate at 16°C for 30 min. 3. Ethanol precipitate by adding 0.1 vol2MNaCL2.5 vol 100% ethanol, and 1 pL glycogen (5 pg/pL) as carrier. Wash with 70% ethanol. 4. Air dry the pellet and reconstitute with 5 pL water. Use 2.5 pL to transform 50 pL of electrocompetent E colr cells (electrocompetence of about 108) 5. Thaw the cells on ice. Pipet 2.5 pL of the ligation mix into the cells and mix gently. Place in an electroporation cuvet and transform by electroporation (machine setting as recommended by manufacturer). 6. Add 1 mL of SOC medium to each vial. Transfer the diluted transformatton mix into a 15-mL plastic tube. Incubate cells at 37°C for 1 h with shaking (250 rpm). 7. Plate 50 and 500 pL from each transformation tube onto LB plates containing the appropriate antibiotic. Grow overnight at 37’C. 8. Pick several tmnsformants and analyze for the presence and orientation of insert by restriction mapping and PCR with nested primers. PCR can be carried out directly on colonies if desired. An alternative way to screen for positive clones is by colony hybridization with labeled oligonucleotides whose sequences are known to be present within the clone. The ultimate check for the veracity of the clone is by sequencing. 9. Grow positive clones and make plasmid preps using standard protocols (e.g., Manratis, Qiagen plasmid kit [see Note 71).

3.6. lntranuclear

Injection

This section is of necessity idiosyncratic, as we use an electrophysiological rig for our injections and to record our functional responses. A full recipe for setting up an electrophysiology rig is beyond the scope of this chapter. However, there is no reason why similar procedures may not be used with commercially available injectors (e.g., Eppendorf). We plate our cells on custom-made microwells, which are based on 22 x 22-mm glass cover slips. We culture our cells on glass to permit the use of acetone-fixation when staining for G proteins (we find that this gives the lowest background). Also, the thin cover slip permits use of high numerical

aperture lenses (with short working

distances) with

the inverted microscope we use for electrophysiology (see Note 8). This improves optical resolution, which aids intranuclear injection. The walls of the well are formed by a 22 x 22-mm square of 3-mm-thick glass, which has had a 15mm diameter hole cut in the center. This is then fixed to the cover slip with a smear of high-vacuum grease. The underside of the cover slip is marked (solvent-based marker) with a grid of 1 x l-mm squares, which are helpful in relocating injected cells. 1. Pull injection electrodes, with a one-stage pull, using a horizontal puller suitable for pulling sharp electrodes. We routinely use a Sutter P97 programmable machine (see Note 9). The settings should be adjusted according to the manufacturer’s instructions until electrodes having a resistance of 30-70 MR, and a relatively short shank (no more than 5 mm) are produced.

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2 Make about 50 pL of a 400~pg/mL solution of plasmid, containing 0.5% FITCdextran, in Ca2+-free Krebs solution (or other extracellular solution). Filter through a 0.2~pm microcentrifuge filter (see Notes 10 and 11). 3. Load 2 pL plasmid solution into the back of the electrode with a 5-pL Hamiltontype gas-tight syringe. Shake the solution down into the electrode tip. 4. Load the electrode onto the holder (see Note 12), which should have a 20-mL syringe connected to it via the side arm. Lower the electrode into the bath solution, 5 Having selected the neuron you wish to inject, and positioned the electrode above it, steadily lower the electrode until it touches the surface of the neuron above the nucleus. On the voltage recording (see Note l3), this wrll be evident as a negative-going voltage deflection during the hyperpolarizing current pulse. Continue pushing downward until the electrode penetrates the nucleus, which will be apparent as a sudden reduction in the amplitude of the voltage deflection, and a slowing of the kinetics of the voltage response. 6. Switch from normal light to fluorescent illumination, and inject dye by applying gentle pressure with the syringe. 7. Make a note of the location of the cell in the grid squares.

3.7. lmmunocytochemistry Antigen-antibody interactions depend on a variety of conditions, including pH (especially a consideration with monoclonal antibodies), temperature, incubation period, antibody concentratron, ionic strength of solution, and fixation. When starting a study, it is essential to optimize the staining conditions for the antibody-antigen complex involved. 1. Wash cells to remove tissue culture medium or existing salt solution. 2. Fix cells using either acetone (20 min at room temperature), acetone:methanol (1: 1 [v/v]; 1 min at -2O’C; see Note 14), or 2-4% paraformaldehyde in PBS (5 min at room temperature). 3. Wash for 3 x 5 min with TBS (see Note 15). 4. Block nonspecific binding of secondary antibody with 10% serum from species B at room temperature for 30 min. 5. Incubate with primary antibody (time and temperature need to be optimized; see Note 16). 6. Wash for 3 x 5 min with TBS. 7. Incubate with secondary antibody (time and temperature need to be optimized) 8. Wash cells for 3 x 5 min with TBS. 9. Incubate with ABC solution for 30 mm at room temperature. 10. Wash for 3 x 5 min with TBS. 11. Incubate with BCIP/NBT (time and temperature need to be optimized; see Note 17). 12. Wash cells for 3 x 5 min with water. 13. Mount in Vectashield.

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14. View cells under a fluorescence microscope to locate injected cells (we use a Zeiss Axiophot) and observe alkaline phosphatase staining under light field (see Notes 1g-20). 15. Photograph cells under fluorescence and light field. 4. Notes 1. Members of G protein families are highly homologous polypeptides. This poses a problem in designing antisense constructs that are specific for only one protein. We have circumvented this problem by making constructs designed against sequences m the 3’ untranslated region, where there is no significant homology among these proteins. 2. The choice of tissue source for the RNA depends on the gene being cloned. The tissue should express the target gene in high abundance. It is also advantageous if the tissue source is easy to isolate from other surroundmg tissues. 3. If the RNA had not been DNase-treated, then dissolve 2 pg RNA in water and adjust the volumes accordingly. 4. The annealing temperature for PCR may be changed, depending on the kind of primers being used. It IS always a good idea to include a positive and a negative control in the PCR amplification step. 5. It is advisable that the TA cloning procedure be done soon after the PCR amphfication step. The TA cloning system makes use of the smgle 3’ A-overhang added by Taq polymerase to each end of the PCR product to enable direct ligation to a linearized vector with 3’ T-overhangs. Through time, the 3’ A-overhangs in PCR products may get degraded. 6. The Amersham DNA ligation system comes with Its own protocol and notes. Volumes for solutions A and B may be adjusted accordingly. 7. For both small and large scale plasmid preparations, we routinely use the Qiagen plasmid kit 8. Visualization of the nucleus of the neuron to be injected is greatly aided by using one of the additional ports of the microscope to set up a video camera, coupled to a monitor. 9. We have used several different micromanipulators to carry out injections, including Narishige hydraulic and mechanical models, and a Sutter version of the Huxley mechanical design. This latter is very much preferred, not so much because it has any advantages for injection, but because it offers superior stability for recordings. 10. We have adopted a strategy to try to minimize Ca*’ loading of the neurons during injection, since we reason that this will be deleterious. We perfuse our cells in the dish with warmed Krebs solution (32’C), as we have noted (S. J. Marsh, personal communication) that the Ca*+ pump of the sympathetic neurons we use as our favored experimental system is impaired at room temperature. We also include 0.5 @4 tetrodotoxin in the external solution, to prevent the neurons firmg action potentials on impalement. We prefer to use Ca*+- containing extracellular solution, because the neurons become detached from the substrate in Ca*+-free medium (even with elevated Mg*+).

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11. Because injected neurons need to be put back into the culture incubator for a sufficient time to allow endogenous protein to run down, precautions need to be taken to prevent infection. We routmely filter our Krebs solution through a 0.2~pm filter, under vacuum, and also wash the perfusion system (reservoirs, tubes, and so on) with 1M HCl, followed by 70% ethanol, followed by filtered Krebs solution. Other precautions mclude washing the microscope stage and perspex bath holder with 70% ethanol. 12. We use an electrode holder designed by David Osborne, of the Pharmacology Department, University College London. The electrode holder can be purchased from the department. This design has several features to ensure that the electrode is held totally rigidly, so that movement of the tip is minimized during application of pressure 13. Detection of contact of electrode with the neuron surface, and of impalement, is assisted by passing hyperpolarizing current into the electrode, once the electrode resistance has been compensated (bridge balance, or equivalent). We will usually use about 0.3 nA of current, although this needs to be varied, depending on the resistance of the electrode. The ObJective is merely to obtain a detectable voltage deflection when the electrode is pushed against the neuron surface. Electrodes can be used to inject more than one neuron, although blocked electrodes (selfevident, as dye is not ejected when pressure is applied) must be discarded. We do not pull many electrodes (usually four or so) in one long run on the electrode puller, since the resultant warmmg-up of the metal filament holder results in sharper (and therefore more easily blocked) electrodes. 14. Acetone:methanol fixation can be done on plastic 15. TBS should be used as the buffer for staining involving alkaline phosphatase, because PBS will inhibit the enzyme. If a nonenzyme-linked fluorescence label is used, PBS may be a more appropriate buffer. 16. Normally, primary antibodies are diluted 1: 100-l : 10,000, with Incubation times ranging from 30 min to overnight, at temperatures ranging from room temperature to 37°C. Secondary antibodies are normally m the same range. 17. The incubation time with BCIP/NBT will depend on the extent of nonspecific staining. We normally use 5 min. 18. If a high nonspecific background is obtained with alkaline phosphatase, try incubating cells with a 10 mMlevamisole solution in final enzyme incubating medium to inhibit endogenous alkaline phosphatase. 19. It is essential to include appropriate controls for the specificity of the antibodies used. Controls for the primary antibody are demonstration of lack of staining in tissue or cells where the antigen is not expressed, and competing out the primary antibody staining by preincubation with the immunogen (for example, the peptide sequence used to raise anti-G protein antibodies). Control for the second antibody by demonstrating the absence of any staining with secondary antibody in the absence of the primary antibody. 20. It is easier to assess changes in the intensity of staining if the microscope is not fully adjusted for phase contrast. We set the phase ring slightly out of alignment for viewing stamed cells.

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References 1, Caulfield, M. P., Jones, S., Vallis, Y., Buckley, N. J., Kim, G.-D., Mulligan, G., and Brown, D. A. (1994) Muscarinic M-current inhibition via Ga,,r I and a-adrenergic inhibition of Ca*+ current via Go, in rat sympathetic neurones. J. Physiol. 477,415-422. 2. Jones, S., Brown, D. A., Milligan,

3. 4.

5. 6.

G., Willer, E., Buckley, N. J., and Caulfield, M. P. (1995) Bradykinin excites rat sympathetic neurons by mhibition of M current through a mechanism involving B2 receptors and Ga,,t t. Neuron 14,39wO5. Hepler, J. R. and Gdman, A. G. (1992) G proteins. Trends Biochem. Scz. 17,383-387. Kleuss, C., Hescheler, J., Ewel, C., Rosenthal, W., Schultz, G., and Wittig, B. (199 1) Assignment of G-protein subtypes to specific receptors inducing inhibition of calcium currents. Nature 353,43-48. Akhtar, S. and Juliano, R. L. (1992) Cellular uptake and intracellular fate of antisense oligonucleotides. Trends Cell Biol 2, 139-144. Wagner, R. (1994) Gene inhibition using antisense oligodeoxynucleotides. Nature

372,333-335. 7. ffrench-Mullen, J. M. H., Plata-Salaman, C. R., Buckley, N. J., and Danks, P. (1994) Muscarinic modulation by a G-protein a-subunit of delayed rectifier K+ current in rat ventromedial hypothalamic neurones. J. Physiol. 474,2 l-26.

17 Protocols Employed of G Protein-Coupled Andrew

in the Investigation Receptor Phosphorylation

6. Tobin

1. Introduction Phosphorylation is a fundamental regulatory mechanism employed by many G protein-coupled receptors (GPCRs). There are presently at least 26 G protein-coupled receptor subtypes that have been demonstrated to undergo agonist-dependent phosphorylation (I). In fact, of the receptor subtypes studied, only three-namely the c&4-adrenoceptor (2), j33-adrenoceptor (3), and the gonadotrophin-releasing hormone receptor ($-are thought not to undergo agonist-sensitive phosphorylation. It seems likely, therefore, that most GPCRs will undergo some form of phosphorylation, and with there being over 100 known members of this gene superfamily, interest in identification of receptor phosphorylation events will remain high. Studies on P-adrenoceptor phosphorylation have centered on chromatographic techniques that have allowed for the purification of the receptor. These techniques have been used to isolate the phosphorylated form of the receptor from cell lines prelabeled with [32P]-orthophosphate (5,, and to obtain large amounts of pure receptor for reconstitution studies. This latter approach has been used extensively in the identification and characterization of P-adrenergic receptor kinase (P-ARK) (6) and has set the benchmark for studies in this field. Indeed, many of the early examples of GPCR phosphorylation required purification of the receptors from intact tissues (7) or from cell lines (8). However, the purification of GPCRs is generally an extremely difficult process. First, the GPCRs receptors are present at very low levels and therefore represent a minor proportion of membrane-bound proteins. Second, many of the receptor subtypes are difficult to solubilize in a way that maintains the ability of the receptor to bind radioligands that are used to monitor the purification. From

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Finally, the purification protocol often involves developing specific affinity chromatography matrices comprising immobilized ligands. These constraints greatly restricted early investigations on GPCR phosphorylation. More recently, many of the problems associated with purification of GPCRs have been overcome by the use of specific antisera that are able to immunoprecipitate receptors following detergent solubilization. This approach has been successfully applied in our laboratory in the study of m3-muscarinic receptor phosphorylation (9). In fact, widespread application of this technique over the past 4 yr has been directly responsible for the rapidly increasing understanding of the diversity of receptor subtypes that undergo phosphorylation (see Note 1). This chapter will outline the protocols we have developed for investigating phosphorylation of the m3-muscarinic receptor in transfected cell lines, using immunoprecipitation. We have also been able to investigate m3-muscarinic receptor phosphorylation in a crude membrane fraction from Chinese hamster ovary (CHO) cells expressing the recombinant human m3-muscarinic receptor (10). These studies have not only demonstrated that the native m3-receptor kinase is at least partly membrane-bound, but also offered the opportunity to investigate the effects of cell impermeable kinase inhibitors in the characterization of the kinase responsible for m3-muscarinic receptor phosphorylation (10). The use of membrane preparations to investigate receptor phosphorylation has also been reported for the A3-adenosine receptor (11) and may in the future be generally applied in studies of a variety of GPCRs. Here, the protocols associated with this technique will be considered, as they pertain to m3-muscarinic receptors. Finally, there have been an increasing number of reports m the literature of the use of bacterial fusion proteins expressing regions of GPCRs as substrates for purified protein kinases or kinases present in cell extracts (see refs. 12 and 23 for examples). Bacterial fusion proteins can be produced and purified in large quantities, and, when used as kinase substrates,can provide a useful indicator as to the likelihood that the intact receptor will act as a kinase substrate, as well as providing an assaythat may be used in the identification of novel GPCR kinases. Here is outlined the protocol we have developed for investigating the phosphorylation of a fusion protein encoding a region of the third intracellular loop of the m3-muscarinic receptor by kinases present in cellular extracts.

2. Materials 2.1. Identification

of GPCR Phosphorylation

in Cultured

Cells

1. HEPES buffered saline (HBS): 10 mA4 HEPES, 0.9% NaCl, pH 7 4. 2. Phosphate-free KrebsIHEPES buffer- 10 mA4HEPES, 118 mA4NaC1,4.3 mi14KC1, 1.17 mA4 MgS04, 1.3 mA4 CaCl*, 25 mA4NaHC03, 11 7 mA4 glucose, pH 7.4.

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3. [32P]-orthophosphate(10 mCi/mL) from Amersham(cat. no. PBS 11). 4. Proteaseinhibitors: 1rmI4phenylmethylsulfonylfluoride (PMSF), 10pg/mL soybean trypsin inhibitor, 1 &mL leupeptin, 1 ccg/mLpepstatin A, 100 pg/mL benzamidine, 100 clg/mLiodoacetamide(seeNote 1). 5 Phosphataseinhibitors: 0.2 mA4sodium vanadate, 2 nnI4 disodium nitrophenyl phosphate,25 mA4glycerophosphate,pH 7.4. 6. Solubilization buffer: 10 rmI4Tris-HCl, 10 mM EDTA, 500 rnA4NaCl, 1% NP40,O.1% SDS,0.5% deoxycholate,pH 7.4 (optional: proteaseinhrbitors + phosphataseinhibitors [seeNote 11). 7. m3-Muscarinic antiserum(332) has beenprevtously characterizedand shownto be specific to the humanm3-muscarinicreceptor in Westernblots and mnnunoprecipitation (9). 8. Protein A-Sepharose slurry: protein A-Sepharose CL-4B (Pharmacia), 1.5 g resuspendedm 50 mL of TE-buffer. 9. TE buffer: 10 mM Tris-HCl, 10mMEDTA, pH 7.4 2.2. Identification of GPCR Phosphorylation in a Crude Membrane Preparation Kinase buffer: 20 mM Tris-HCl, 10 mI4 MgClz,l mM EGTA, 2 miI4 DTT (dithiothreitol), pH 7.4. 2.3. Phosphorylation of a Bacterial Fusion Protein Encoding a Region of the m3-Receptor Glutathionc-Sepharose slurry: 10 mL of glutathione-Sepharose resuspended in 40 mL of TE buffer.

beads

3. Methods 3.1. Identification

of GPCR Phosphorylation

in Cultured

Cells

The protocol outlined below involves identification of the phosphorylated form of the human m3-muscat-uric receptor expressed in a transfected CHO cell line (CHO-m3 cells; expressing -1.3 pmol receptor/mg protein). This mvolves three distinct steps: labeling of the intracellular ATP pool with [32P]P,; stimulation of the cells, followed by solubilization of the receptor; and immunoprecipitation of the solubilized receptor. The protocol utilizes a receptor-specific anttserum raised against the third intracellular loop of the m3-muscarinic receptor (9). There are, however, a number of reports in the literature in

which the receptorsexpressedasrecombinantproteins in transfectedcell lines have been tagged with an epitope tag (see Note 2). This allows for immunoprecipitation of the receptor with commercially available antiserum against the epitope tag.

Tobin

230 3.1.1. p2P]-Labeling

of Cellular ATP Pool and Receptor Solubilization

1. Harvest CHO-m3 cells using HBUO.5 mMEDTA and wash twice m phosphatefree Krebs/HEPES buffer. 2. Resuspend the cells in phosphate-free Krebs/HEPES buffer at a density of 1-3 x 1O6cells/n& and aliquot into 1-mL aliquots (see Note 3). 3. Add [32P]-orthophosphate (50 pCr/nL) and incubate the cells at 37°C for 60 mm (see Note 4). 4. The cell suspensions can now be challenged with experimental reagents (e.g., 1 mA4 muscarmic agonist for O-15 min). Terminate the stimulation by rapid centrifugation (-2000 rpm on bench top Microfuge for 30 s), aspiration of medium, and addition of 1 mL of ice-cold solubilization buffer. 5. Solubilize the m3-muscarinic receptors on ice for 30 min and then clear the sample by centrifugation in a Microfuge (maximum speed for 3 mm). Save the supernatant for immunoprecipitation of the solubilized receptor (see Section 3.1.2.).

3.1.2. Receptor lmmunoprecipitation 1. Add 3 J.L of protein A-purified m3-muscarmtc receptor antiserum (332) (9) to the solubilized cell extract (in Section 3.1.1.) and incubate on me for 60-90 min. 2. Add 175 pL of protein A-Sepharose slurry to the sample and set rotating for 15 mm at room temperature. 3 Pellet the protein A-Sepharose by brief centrtfugatron (maximum speed m a Microfuge for 30 s), aspirate the supernatant, and wash the pellet 3-5 times wtth TE buffer. 4. At the final wash step, remove as much of the supernatant as possible by using a fine pipet tip. 5 Resuspend the pellet in 20 pL of 2X SDS PAGE sample buffer. Heat the sample to -85°C for exactly 2 min (see Note 5) and then load onto an 8% SDS-PAGE gel to resolve the proteins. 6. Stain the gel with 0.2% Coomassre blue m 50% methanol/lO% acetic acid, and destained in 50% methanol/lO% acetic acid. This staining procedure will visualize the immunoprecipttated antibody, thereby confirmmg equal rmmunoprectprtation/loading; however, the receptor will not be visible because of the low quantity. Dry the gel and obtain an autoradiograph.

3.2. Identification of GPCR Phosphorylation in a Crude Membrane Preparation Although it is extremely desirable to demonstrate receptor phosphorylation in an intact cell, only limited characterization of the kinase can be carried out because of the restriction of being able to use only those inhibitors and reagents

that have the ability to cross the plasma membrane. During our work on the muscarinic receptor kinase, we were able to demonstrate that the membrane fraction from CHO-m3 cells contained a native kmase that was able to phosphorylate

the m3-muscarinic

receptor in an agomst-dependent

manner

(IO).

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Protocols

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Using this preparation, we were able to introduce cell-impermeable reagents designed to inhibit/activate the native muscarinic receptor kinase (IO), and also, in later studies, to use the membrane preparation in reconstitution experiments with a purified protein kinase to investigate the ability of exogenously added kinase to increase the agonist-sensitive phosphorylation of the intact m3-muscarinic receptor (22). The technique can be divided into three distinct sections: preparation of the membrane fraction, stimulation of receptor phosphorylation, and solubilization and immunoprecipitation of the receptor. 3.2.7. Preparation of a Crude CHO-m3 Membrane Fraction 1. Harvest CHO-m3 cells using HBUO.5 mM EDTA (use at least two confluent 175-cm* flasks) and resuspend the cell pellet in ice-cold TE buffer (15 mL), plus

proteaseinhibitors. 2. Leave cells on ice for 10 min and then disrupt in a tissue homogenizer using one 15-s pulse. 3. Remove cell debris by centrifugation at 2000g for 3 min. Collect the supernatant that contains the membrane fraction and pellet the membranes by centrifugation at 15,OOOg for 10 mm. 4. Resuspend membrane pellet in kinase buffer plus protease inhibitors, adjusting the protein concentration to 1 mg protein/ml.

3.2.2. Stimulation of m3-Muscarinic Receptor Phosphorylation 1. To 50 pL of the above membrane preparation (-0.1 pmol of receptor), add stimulatory reagents (e.g., the muscarinic agonist methacholine) and/or exogenous protein kinase preparation or buffer blank. Add kinase buffer to bring the final volume to 100 pL and start the reaction by adding 100 @4 [Y-~*P] ATP (l-4 cpm/fmol ATP). 2. Continue the reaction at 32’C for the required time (e.g., 10 mm; see Note 6). 3. Stop the reaction by either directly adding 1 mL ice-cold solubrhzatton buffer or by first pelleting membranes by a brief centrifugation (30 s) m a microfuge (maximum setting), followed by aspiration of the supematant and resuspension of the pellet in 1 mL ice-cold solubilization buffer. 4. Solubihze the m3-muscarinic receptor on ice for 30 min. Clear the supematant by centrifugation (13,OOOg, 3 min) and immunoprecipitate the solubilized m3muscarinic receptor, as described in Section 3.1.2. Resolve the immunoprecipltated proteins on an 8% SDS-PAGE gel. Stain and destain the gel (as outlined in Section 3.1.2.) before drying and obtaining an autoradiograph.

3.3. Phosphorylation of a Bacterial Fusion Protein Encoding a Region of the m3-Muscarinic Receptor Bacterial fusion proteins consisting of a domain, usually at the N-terminus, that allows for easy purification of the fusion protein, followed by a domain corresponding to the recombinant receptor under investigation, are now widely employed in btochemical experiments. We have produced a fusion protein,

232

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termed Ex-m3, that at the N-terminus encodes glutathione-S-transferase, which allows for purification of the fusion protein on glutathione-Sepharose beads, and at the C-terminus a region of the third intracellular loop of the m3-muscarinic receptor (9). The vector construct consists of a pGEX-2T (Pharmacia) backbone in which the m3-muscarinic receptor sequence encoding amino acids Ser345-Leu463 was cloned downstream and inframe with the glutathione-S-transferase sequence (see Note 7). Production of the fusion protein encoded m this vector can be induced from transformed bacteria by addition of 0.1 mA4isopropyl-fi-n-thiogalactopyranoside (IPTG). The fusion protein can then be purified from abacterial lysateusing a glutathione-sepharosematrix (Pharmacia). The protocol described below outlines the use of the purified fusion protein, Ex-m3, in an in vitro assay to identify the presence of a kinase extracted from CHO cells. 1. Preparea high-speed supematant extract from CHO cells by first harvestmg CHO

2.

3

4. 5.

6 7.

8

cells using HBWO.5 mM EDTA (use at least three confluent 175cm2 flasks) Resuspend the cell pellet in 2 mL kmase buffer plus protease inhrbttors and leave to swell on ice for 10 mm. Disrupt the cells with a 10-s pulse in a tissue homogenizer and then spm the sample at 300,OOOg in a benchtop ultracentrifuge for 30 mm. Collect the supernatant and adjust the protein concentration to l-5 mg protem/mL. Take 10 pL of this cytosohc extract and add 3.5 pg of fusion protem, Ex-m3 Adjust the volume to 100 pL with kmase buffer and start the reaction by adding 50 fl [Y-~~P] ATP (0 4-l 0 cpm/fmol ATP) Continue the mcubatton at 37’C for 10 mm Stop the reaction by adding 1 mL of ice-cold TE buffer. Extract the fusion protein by adding 200 pL of a glutathione-Sepharose slurry. Collect the glutathione-Sepharose beads by a brief centrtfugation m a microfuge (maximum setting for 30 s), Aspu-ate supematant and wash the pellet three times m ice-cold TE buffer. At the final wash step, remove as much of the supematant as possible by using a fine pipet tip. Resuspend the pellet in 20 pL of 2X SDS PAGE sample buffer. Denature the sample in boiling water for 3 min and then load on a 12% SDS-PAGE gel to resolve the proteins. To visualize the fusion proteins and confirm their posmon on the gel, stain the gel with 0.2% Coomassie blue in 50% methanol/lO% acetic acrd, and destained in 50% methanol/lO% acetic acid. Dry the gel and obtain an autoradiograph.

4. Notes 1 PMSF, benzamidine, and iodoacetamide undergo hydrolyses, and thereby inactivation, in aqueous solutton. PMSF 1smade up in a 200~mM stock in ethanol and the benzamidine as a lOO-mg/mL stock in ethanol. These reagents are stable in ethanol and should be added to the buffers at the very last moment. Iodoacetamide is prepared in aqueous solution (usually at a stock concentration of 100 mg/mL)

GPCR Phosphorylation Protocols

2.

3.

4. 5.

6.

7.

233

and added immediately to the buffer just prior to use. This author has carried out many experiments in which the protease and phosphatase inhibitors have been omitted from the solubilizatron buffer with no apparent effect on the phosphorylation results. It would appear that the harsh condittons of the solubilization buffer are sufficient to inhibit phosphatase and protease activity, although one may want to include the inhibitors. An alternative approach to raising receptor subtype-specific antibodies for immunoprecipitation studies is to epitope tag the receptor, usually at the N-terminus. The solubihzed receptor can then be immunoprecipitated using commercially available monoclonal antibodies raised against the epitope tag, One epitope tag employed in GPCR studies is the influenza hemagglutinin (HA) tag YPYDVPDYA, which is recognized by the 12CA5 monoclonal antibody available from BabCo, Berkeley Antibody, and Boehringer Mannhelm. A shorter HA tag, DVPDYA, has also been reportedly recogmzed by the 12CA5 antibody. Also, the FLAG tag DYKDDDDK, which is recognized by the FLAGM2 monoclonal antibody available from Kodak IBI, is commonly used. Note that the commercial anti-epitope tag antibodies may be mouse monoclonal antibodies that have loweraffinity binding to protein A than rabbit antibodies (see ref. 14). It may, therefore, be necessary to Include an extra step in the rmmunoprecrpitation protocol. Following mcubation wrth the primary antrserum (antiepitope tag antiserum) an antimouse IgG antibody raised in the rabbit should be added. This will form a receptor:mouse antiepitope tag IgG: rabbit antimouse IgG complex that will have a high affinity for protein A-Sepharose beads. If the cell density is too high, then after solubilization the cellular DNA forms into a stringy mass. If this occurs, it can severely interfere with the immunoprecipitation. The remedy is either to use fewer cells or break up the DNA by vortexing and rapid pipeting through a narrow bore pipet tip before addition of the antireceptor antiserum. We have calculated the specific activity of the intracellular ATP pool following this labeling protocol to be 840 f 16 1 cpm/pmol ATP. Because of the hydrophobic nature of GPCRs when denatured by boiling in SDSPAGE buffer, the receptors can form large aggregates that remain trapped at the top of the gel. We overcome theseproblems by denaturing the sample at 85°C for 2 min. Using this approach nearly all of the m3-receptor is resolved wtthm the SDS-PAGE gel. It is important to remember that the membrane preparation is very crude and will contain a considerable amount of ATPase activity. We have calculated that -80% of the ATP is consumed within 10 min. This is important to bear in mind when interpreting the time-course data for phosphorylation of the receptor. Detailed protocols for cloning into the pGEX vector system, transformation of bacteria, induction of fusion protein, and purification of fusion proteins can be obtained from Pharmacia.

Acknowledgment I thank the Wellcome

Trust for financial

support.

Tobin

234

References 1. Tobin, A. B. (1996) Receptor-specific kinases in the regulation of phospholipase C-coupled receptors, m The Phospholipase C pathway Its Regulation and Desensitisatzon (Tobin, A. B., ed.), Landes, Austin, Texas. 2. Kurose, H. and Lefkowitz, R. J. (1994) Differential desensitisatlon and phosphorylation of three cloned and transfected az-adrenergtc receptor subtypes. J. Bzol Chem 269, 10,093-l 0,099. 3. Liggett, S. B. and Lefkowrtz, R. J (1994) Adrenerglc receptor-coupled adenylyl cyclase systems: regulation of receptor function by phosphorylation, sequestration and down-regulation, in Regulation of Cellular Signal Transduction Pathways by Desensitrsation and Amphjkatron (Sibley, D. R and Houslay, M D., eds.), John Wiley, Chichester. 4. McArdle, C. A., Forrest-Owen, W , Willars, G , Davidson, J , Poch, A., and Kratzmeier, M. (1995) Desensitisation of gonadotropin-releasing hormone action in the gonadotrope derived aT3- 1 cell line. Endocrrnology 136,4864-487 1. 5. Stadel, J. M., Nambt, P., Shorr, R. G. L., Sawyer, D. F., Caron, M. G., and Lefkowitz, R. J. (1983) Catecholamme-induced desensitisatlon of turkey erythrocyte adenylate cyclase IS associated with phosphorylatlon of the j3-adrenergic receptor. Proc. Natl. Acad. Sci. USA SO,3 173-3 177. 6 Benovrc, J. F., Mayor, F., Staniszewski, C., Letkowttz, R. J., and Caron, M. G. (1987) Purification and characterisation of S-adrenergic receptor kinase. J Biol Chem. 262,9026-9032.

7. Kwatra, M. M. and Hosey, M. M (1986) Phosphorylation inic receptor in intact chick heart and its regulation by J Biol. Chem. 261, 12,429-12,432. 8. Leeb-Lundberg, L. M., Cotecchia, S., DeBlasi, A., Caron, R. J. (1987) Regulation of adrenergic receptor function

of the cardiac muscara muscarinic agonist M. G., and Lefkownz, by phosphorylatron.

J. Biol. Chem. 262,3098-3105.

9. Tobin, A. B. and Nahorskt, S. R (1993) Rapid agonist mediated phosphorylation of m3muscarinic receptors revealed by immunoprecipitatron. J. Biol. Chem. 268,98 17-9823. 10. Tobin A. B., Keys B., and Nahorski S. R. (1993). Phosphorylation of a phosphoinositidase C-linked muscarinic receptor by a novel kmase distinct from /3-adrenergic receptor kinase. FEBS Lett. 335,353-357. 11. Palmer, T. M., Benovic, J. L., and Stiles, G L. (1995) Agonist-dependent phosphorylation and desensitisation of the rat As-adenosme receptor. J. Biol Chem. 270,29,607-29,6 13. 12. Tobin, A. B., Keys, B., and Nahorski, S. R. (1996) Purificatton and characterisation of a novel receptor-specific kinase that phosphorylates a PLC-coupled muscarinic receptor. J Biol Chem 271, 3907-3916. 13. Prossnitz, E. R., Kim, C. M., Benovic, B. L., and Ye, R. D. (1995) Phosphorylation of the N-formyl peptide receptor carboxyl terminus by the G protein coupled receptor kinase, GRK2. J. Biol Chem 270, 1130-l 137. 14. Harlow, E. and Lane, D. (1984) Antrbodies: A Laboratory Manual. Cold Sprmg Harbor Laboratory, Cold Spring Harbor, NY.

18 Assay of G Protein-Coupled Receptor Kinase Activity by Rhodopsin Phosphorylation Alison W. Gagnon and Eamonn Kelly 1. Introduction Agonist occupation of G protein-coupled receptors (GPCRs) leads to a cellular response that wanes, or desensitizes, with prolonged agonist exposure. Different cellular mechanisms contribute to desensitization, including the altered function and/or expression of receptors, G proteins, and effecters. Phosphorylation plays a major role in the rapid (seconds to minutes) desensitization of responses generated by these receptors (I). Apart from phosphorylation by second messenger-dependent kinases, such as cyclic AMP-dependent protein kinase and protein kinase C, a family of protem kinases has been identified whose specific function appears to be the phosphorylation of GPCRs, leading to their uncoupling from the G protein and hence loss of further responsiveness (reviewed in refs. 2 and 3). These kinases have been termed G protein-coupled receptor kinases (GRKs); the mammahan family of GRKs presently consists of six members, termed GRKl-GRK6. These include GRKl (also known as rhodopsin kinase), which phosphorylates light-activated rhodopsin, the GPCR for light in rod cells of the retina, GRKs 2 and 3 (also known as P-ARK1 and P-ARK& for their ability to phosphorylate agonist-occupied P-adrenoceptors), and GRKs 4, 5, and 6. On the basis of sequence homology and functional properties, GRKs can be subdivided into three groups, the first containing GRKl, the second GRKs 2 and 3, and the third GRKs 4, 5, and 6. A major functional distinction between GRK2 and 3 and the others is that the activity of the former is greatly enhanced by G protein /3r subunits, but that of GRKs 1,4,5, and 6 is not (4). Apart from GRKl (retina) and GRK4 (testes), the distribution of the other GRKs is wtdespread. In addition, the GRKs are able to phosphorylate, at least in vitro, a number of different From

Methods in Molecular Brology, vol 83 Receptor SIgnal Transductron Edtted by R A J Chalks Humana Press Inc , Totowa, NJ

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Protocols

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G protein-coupled receptor substrates. For example, GRK2 can phosphorylate agonist-occupied &adrenoceptors, a2-adrenoceptors, substance P, Al adenosine, and M2 muscarinic receptors, as well as light-activated rhodopsin. The selectivity of GRKs for particular receptors in vivo remains to be determined, although initial studies indicate that selectivrty does exist (5,6). The ability of GRKs to phosphorylate light-activated rhodopsin is particularly useful and it forms the basis of a simple assay to assessthe activity of a GRK preparation (GRK4 has not been tested with rhodopsin, but can phosphorylate the purified P2-adrenoceptor; see ref. 7). The basic assay consrsts of incubation of the kinase preparatton with rod outer segments (ROS) in the presence of [32P]-ATP and light. Phosphorylated rhodopsin is then separated from free [32P]-ATP and other phosphorylated proteins by gel electrophoresis and the degree of rhodopsin phosphorylation is assessedby autoradrography and scintillation counting. Rhodopsin is prepared as rod outer segments, as first described by Wilden and Kuhn (8) and Schichi and Somers (9), where it constitutes greater than 90% of the expressed protein. This assay is ideal for purified GRK preparations or for cells in which a particular GRK has been overexpressed, but it can also be used to assessGRK activity in crude cell extracts, provided that the level of GRK activity is not too low.

2. Materials 2.7. Preparation of Urea-Treated Bovine Rod Outer Segments 1. Dark-adapted frozen bovme retinas (can be obtained from Rockvtlle Rockville, MO). 2. Homogenization buffer: Add 68 g sucrose to 132 mL of 65 miVNaC1, MgC12, 10 mMTris-acetate, pH 7.4. Store at 4°C

Meat, 2 miI4

3. 10 mMTris-acetate, pH 7.4 (500 mL, store at 4°C). 4. 10 nuI4 Trts-acetate, pH 7.4, 1 mM MgClz (1000 mL, store at 4°C). 5. Sucrose buffers (store at 4V): a. 0.77M sucrose (200 mL): bring 52.7 g sucrose to 200 mL with 10 n&f Trisacetate, pH 7.4, 1 mA4 MgC12; b. 0.84M sucrose (100 mL): bring 28.2 g sucrose to 100 mL with 10 miI4 Tns-

acetate,pH 7.4, 1 mMMgC12; c. l.OOA4 sucrose (100 mL): bring 34.2 g sucrose to 100 mL wtth 10 miW Trisacetate pH 7.4, 1 mit4 MgCl,. 50 mMTris-HCl, pH 7.4, 5 mMEDTA, 5Murea (100 mL, store at 4“C). 50 mikf Trts-HCl, pH 7.4 (1000 mL, store at 4°C). 20.5-gage needle and syringe. Sucrose needle. Sonicator.

6. 7. 8. 9. 10. 11. 40 mL Wheatonhomogenrzer,A pestle.

Rhodopsin Phosphorylation

Assay of GRKs

237

12. Safelight 13. Centrifuge rotors: SS34, SW27, Ti60, or equivalent.

2.2. Phosphorylation 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

of Urea-Treated

Rod Outer Segments

20/2 Buffer 20 mA4 Tris-HCl, pH 7 5,2 mM EDTA. lMMgC1,. 10 mM ATP (frozen in small aliquots at -2O’C). Purified preparations of P-ARK or crude cell preparation (see Notes 1 and 2). [Y-~~P]-ATP (NEN Du Pont, Boston, MA, NEG-002A, 10 mCi/mL) Urea-treated ROS Thermostatically controlled water bath set to 30°C. Rack with sufficient spaces to hold all assay tubes m 30°C water bath. 100 miV sodium phosphate, pH 7.0,s mA4EDTA. Gel electrophoresis kit (e.g., Bio-Rad mini-Protean II [Hercules, CA]). Prestained mol wt standards (e.g., Bio-Rad cat no. 161-0305). 3X SDS-sample buffer: 8% SDS, 50 mil4 Tris-HCl, pH 6.5, 10% glycerol, 5% mercaptoethanol, and 0.005% bromophenol blue; store at -2O’C. Coomassie blue solution, Dissolve 1 g Coomassie blue R-250 m 300 mL methanol, add 100 mL acetic acid and 600 mL distilled water. Destain solution: 300 mL methanol, 100 mL acetic acid, 600 mL distilled water. Whatman 3MM tilter paper Gel dryer (e.g., Bio-Rad model 543 gel dryer). Autoradiograph cassettes and X-ray film (e.g., FUJI medical X-ray film, FisherBiotech autoradiography cassette FBAC 8 10, Fisher Scientific, Pittsburgh, PA). Scmtillation vials.

3. Methods 3.1. Preparation of Urea-Treated Bovine Rod Outer Segments Important: All the following steps should be carried out m the dark under safelight illumination. 1. Thaw 100 frozen dark-adapted bovine retinas in 100 mL of me-cold homogenization buffer in a 200 mL conical flask. Keep flask on ice. Once thawed, shake vigorously for 4 min. 2. Centrifuge in Sorval tubes at 2OOOgfor 4 min at 4°C (4000 rpm in an SS34 rotor), 3. Carefully remove supematant with a sucrose needle. 4. Add 200 mL of 10 mMTris-acetate, pH 7.4, buffer to supematant. Centrifuge at 43,000g for 4 min at 4°C (19,000 rpm in a SS34 rotor). Retain the orange pellet. If the supematant is strongly orange, dilute with additional 10 mM Tris-acetate, pH 7.4, buffer, and recentrifuge. 5. Resuspend the pellets in a total of -30 mL of 0.77M sucrose buffer. 6. Dounce homogenize on ice in a 40-n& Wheaton Dounce homogenizer (A pestle), with 15 vigorous strokes. Draw the homogenate through a 20.5-gage needle 5-10 times, followed by further Dounce homogenization with five strokes.

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7. Layer the sample on top of a sucrose gradient prepared in tubes for an SW27 rotor (Sorvall AH-627, 36 mL swinging bucket) or an SW41 rotor (if SW27 is not available): sample in 0 77M sucrose on top; 5 mL 0.84M sucrose (3 mL for SW4 1); and 5 mL 1.OOA4sucrose on bottom (3 mL for SW41) 8 Centrifuge at 115,OOOg for 30 mm at 4°C (26,000 rpm for SW41 rotor). 9. Remove the strong orange band at the 0 84/1.00 interface with a sucrose needle. Dilute the sample with an equal volume of 10 mM Trts-acetate, pH 7.4, 1 mM MgCI, and centrifuge at 43,000g for 20 mm (19,000 rpm m an SS34 rotor). The pellets can be stored at -80°C at this point, tf desired (wrapped in aluminum fotl). 10. Resuspend the pellets in 25 mL of a solution of 50 mM Tris-HCl, pH 7.4,5 mM EDTA, 5M Urea. 11. To denature the endogenous kmases, sonicate the sample on high power for 4 mm m a tube immersed m ice water (this also needs to be done in a darkened room using a safelight). 12. Add -50 mL 50 mM Trts-HCI, pH 7 4, buffer to the sample and centrifuge m an ultracentrifuge at 204,OOOg for 45 min at 4°C (45,000 rpm m a Ti60 rotor). 13. Resuspend the pellets m -5 mL 50 mMTris-HCl, pH 7.4, by vortexing or mtld homogenizatton with a polytron. Fill the tubes with 50 mMTris-HCl, pH 7.4, and then recentrifuge (see step 12). 14. Repeat this washing step two additional times. 15. Resuspend the final pellet in a total volume of 5 mL of 50 m/t4 Tris-HCl, pH 7.4, by using a polytron on low speed and then draw the sample through a 20.5-gage needle and syringe several times. 16. Make small aliquots of the urea-treated ROS in Eppendorf tubes (25-50 pL) wrapped m aluminum foil and freeze m liquid nitrogen. Store at -80°C. Use one ahquot to check the color (it should be red) and for protein determmation. This preparation typically yields 10-25 mg of rhodopsm from 100 retinas.

3.2. Phosphorylation

of Urea-Treated

Rod Outer Segments

1. Make a sufficient volume of assay buffer for all samples. For 20 pL total assay vol, 1 mL of assay buffer will be sufficient. For 1 mL assay buffer, add 5 pL of lMMgCl* (final concentration is 5 mMMgCl& to 995 pL of 20/2 buffer. 2. If using purified preparations of P-ARK, dilute P-ARK so that the final concentration m assay tube is 10-50 nA4 (see Notes 1 and 9). 3. Crude cell extracts should be prepared as m Note 2. It is best to initially use several concentrations of lysate, smce the level of expression and acttvrty of the kinase is probably not known. 4. Prepare ATP solution. For each reaction use -1 pCt [T-~~P]-ATP. For 10 reactions, add 10 pCi [Y-~~P]-ATP (see Note 4), 2 pL 10 mA4ATP, and enough assay buffer to bring total volume up to 20 pL. For quantitation of incorporation, count duphcate 2 pL aliquots of ATP solution m a scmtillation counter. The cpm may exceed the capacity of the mstrument; tf this 1s the case, count a dilution of the ATP solution.

Rhodopsin Phosphorylation Assay of GRKs

239

5. Make a dilution of urea-treated rod outer segments (ROS) in assay buffer so that the final amount m each assay tube will be 0.5 pA4 ROS (i.e., 0.4 l.rg ROS per tube, but see Notes 3 and 9). Perform this step in a dark room under safe light illumination. Wrap tube containing the appropriate dilution of ROS in aluminum foil and leave on ice until ready to add to final reaction mix. The stock tube of urea-treated ROS can be refrozen at -70°C several times. 6. A typical 20-pL assay can then be set up on ice in an Eppendorf tube as follows: a. -16 ng of purified P-ARK diluted as in Note 1 (for final concentration of 10 nM), or l-3 pL crude cell lysate; b. Assay buffer so that final volume m tube will be 20 &; c. 2 pL of ATP solution prepared in step 4; and d. In a dark room under safe light illumination, or m very low light, add 0.4 ~18 urea-treated ROS. Two controls should be run. The first is a single tube without P-ARK or crude cell lysate to determine background phosphorylation. The second is to have a set of tubes incubated in the dark wrapped in aluminium foil before placing in the water bath to determme agonist dependency of phosphorylation (see Note 5). 7. Incubate reaction tubes in a 30°C water bath under constant room light illumination for an appropriate amount of time. For experiments m which quantitation IS not necessary, 20-30 min is sufficient. When comparison of the level of phosphorylation is important, especially between samples, shorter mcubation times may be necessary (see Note 6). 8. Stop reaction by the addition of 10 pL 3X SDS sample buffer. If background phosphorylation of cytosolic proteins present in the lysate is a problem for crude kinase preparations, it may be preferable to stop the reaction with the addition of 1 mL of ice-cold 100 mM sodium phosphate, pH 7.0, 5 mM EDTA buffer, followed by centrifugation in a microcentrifuge. Resuspend the ROS pellets in a final concentration of 1X SDS sample buffer for electrophoresis (sonication may help resuspension). Irrespective of which method is used to stop the reaction, it is important to incubate for at least 30 min in SDS sample buffer before gel electrophoresis (if not denatured with a high concentration of SDS, rhodopsm can dimerize and give multiple bands on SDS-PAGE). 9. Run stopped reactions on a 10% (w/v) polyacrylamide gel (see Note 7). Run gel until dye front has reached the bottom of the gel. 10. Before drying the gel, the dye front should be removed (see Note 8) with a razor blade. At this point the gel can be stained with Coomassie blue solution for - 15 min and then destained with several changes of Destain solution. Dry gel on Whatmann 3MM filter paper. If quantitation is necessary, bands can be excised with a scalpel blade and counted in a scintillation counter (see Note 10).

4. Notes 1. For purified preparations of P-ARK, the kinase must be diluted in 20 miUHEPES, pH 7.5, 5 mM EDTA, O.lMNaCl with 0.02% Triton X- 100. This stabilizes P-ARK and prevents it from sticking to the sides of the tube. Since this buffer has

240

2.

3.

4.

5.

6.

7.

Gagnon and Kelly high concentrations of salt that inhibit P-ARK, it must be diluted at least lo-fold in the assay buffer. Preparation of crude lysates. Make lysate buffer composed of 200 mMNaC1,20 mA4 HEPES, pH 7.5, 10 mM EDTA, 1 mM DTT, 0.02% Triton X-100, 0.2 mg/mL benzamidine, 0.5 mA4PMSF, and 0.02 mg/mL leupeptin. Harvest cells and wash two times in ice-cold phosphate-buffered saline. Add ice-cold lysis buffer to the cell pellet or add directly to adherent cells on a cell culture dish, scrape and put m a tube for homogemzation. Homogenize with a polytron twice for 30-s pulses at near-maximum setting. Spin at maxrmum speed in Eppendorf tube for 10 min. Remove supernatant and put in a fresh tube on ice. Do protein assay and use a fresh preparation for assay. When assaying purified P-ARK preparations, 0.5 @4rhodopsm should be sufficient. Htgher concentrations of rhodopsin (up to 5 pil4) may be preferable for ceil lysate preparations, or when the reaction has been microcentrifuged before addition of SDS sample buffer. When using crude cell lysates, start by adding l-3 pL of the lysate. Crude cell lysates contain many inhibitors of phosphorylation and the use of more lysate can actually decrease the amount of phosphorylation. Results with crude cell lysates may vary, depending on the endogenous level of expression of the various GRKs and their respective activities, but this has been successfully used for human mononuclear leukocytes (10) and we have used it for NG108 15 neuroblastoma x ghoma cells. GRKs have been over-expressed in several cell lines, including COS (11,12) and SF9 (13,14) and crude cell lysates from these cells have also been used successfully in this assay. Check the specific activity and the reference date of the [Y-~~P]-ATP for each shipment. If the specific activity is greater than 10 mCi/mL, adjust with 50 mM HEPES, pH 7.5. The half-life of 32P is 14.3 d. Therefore, the amount of radiolabeled [Y-~~P]-ATP in the stock bottle must be adjusted for decay before use in each assay. Rhodopsin is susceptible to phosphorylation by protein kinase C, so it is important with crude cell lysates to demonstrate that the phosphorylation seen is agonist-dependent. This can be accomplished by keeping one set of samples in the dark. After addition of the urea-treated ROS m the dark, the assay tube can be wrapped in aluminum foil and incubated at 30°C in the rack with all the other samples. When the incubation time is over, assay tubes can be placed on ice and stopped all at the same time in the dark. For experiments in which comparison of the level of urea-treated ROS phosphorylation is desired, it is necessary to ensure that the trme-course of phosphorylation is in the linear range. Therefore, it maybe necessary to run a time-course of phosphorylation for your samples. SDS-PAGE: For one 10% mini gel (e.g., Bio-Rad Mini-Protean II electrophoresis system), mix 1.68 mL 30% acrylamide/0.8% bisacrylamrde; 2.5 mL 0.75M Tris, pH 8.8; 750 pL distilled water; 50 uL 10% SDS; 25 pL 0 5MEDTA; 30 pL 10% ammonium persulfate; and 5 pL TEMED. Immediately after adding TEMED, pipet 3.5 mL into gel apparatus. Overlay with 0.1% SDS and allow

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30-45 min for gel to polymerize. Drain off 0.1% SDS and dry interface with a small piece of Whatman 3MM paper. Make the stacking gel as follows: 150 pL 30% acrylamide/0.8% bisacrylamide; 250 pL 0.75MTris, pH 6.5; 22.5 pL 10% SDS; 1.05 mL distilled water; 25 pL 10% ammonium persulfate; 11.25 pL 0.5M EDTA; and 2.5 pL TEMED. Mix and pipet on top of resolving gel, place comb into stacking gel and allow to polymerize for 10-l 5 min. When polymerization is complete, remove the comb and rinse the wells with distilled water. Run gel in 25 nut4 Tris base, 250 m&f glycine, 0.1% SDS buffer at 180 V for about 1 h or until the dye front reaches the bottom of the gel. 8. After electrophoresis of the SDS-PAGE gel, remember to remove the bottom of the gel to get rid of the free [y-32P]-ATP that runs just ahead of the dye front. This will give a cleaner autoradiograph. 9. For calculating mol wt of the urea-treatedROS, 40 pg = 1 nmol (mol wt = 40,000 g/mol). For preparations of purified P-ARK, use 80 pg = 1 run01 (mol wt = 80,000 g/mol). 10. When counting excised bands and ATP solution for quantitation, both must be counted in the same manner. If scintillant is used for counting ATP solution, then it must be used when counting excised bands. To calculate the stoichiometry of incorporation, divide the input cpm (2 pL of ATP solution counted in step 4, remembering to take into consideration any dilutions made) by the pmol of cold ATP in each assaytube. This gives cpm/pmol ATP. Take the value for each band that was counted and divrde by cpm/pmol ATP. This gives pmol of radiolabeled phosphate incorporated in that band; dividing this value by pmol of urea-treated ROS in each assay results in the stoichiometry of incorporation on a mol to mol basis.

References 1. Hausdorff, W. P., Caron, M. G., and Lefkowitz, R J. (1990) Turning off the signal: desensitization of P-adrenergic receptor function. FASEB J. 4,288 l-2889. 2. Haga, T., Haga, K., and Kameyama, K. (1994) G protein-coupled receptor kinases. J. Neurochem 63,400-4 12. 3. Premont, R. T., Inglese, J., and Lefkowitz, R. J. (1995) Protein kinases that phosphorylate activated G protein-coupled receptors. FASEB J 9, 175-182. 4. Haga, K. and Haga, T. (1990) Dual regulation by G proteins of agonist-dependent phosphorylation of muscarinic acetylcholine receptors. ,FEBS Lett 268,43-47 5. Kong, G., Penn, R., and Benovic, J. L. (1994) A /3-adrenergic receptor kinase ,dominant negative mutant attenuates desensitization of the l&adrenergic receptor. J Biol Chem. 269, 13,084-13,087. 6. Dawson, T. M., Arriza, J. L., Jaworsky, D. E., Borisy, F. F., Attramadal, H., Lefkowitz, R. J., and Ronnett, G. V. (1993) /3-Adrenergrc receptor kmase-2 and P-Arrestinas mediators of odorant-induced desensitization. Science 259,825-829. 7. Premont, R. T., Macrae, A. D., Stoffel, R. H., Chung, N., Pitcher, J. A., Ambrose, C., Inglese, J., MacDonald, M. E., and Lefkowitz, R. J. (1996) Characterization of the G protein-coupled receptor kmase GRK4. J. Bzol. Chem. 271,6403-6410.

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8. Wtlden, U. and Kuhn, H. (1982) Light-dependent phosphorylation of rhodopsin. number of phosphorylation sites. Bzochemtstry 21,3014-3022. 9. Shichi, H. and Somers, R. L. (1978) Light-dependent phosphorylation of rhodopsin. J. Blol Chem. 253,7040-7046. 10. Chuang, T. T., Sallese, M., Ambrosini, G., Parruti, G., and De Blasi, A (1992) High expression of S-adrenergic receptor kinase in human peripheral blood leukocytes. J. Biol. Chem. 267,6886-6892. 11. Benovic, J. L., DeBlasr, A., Stone, W. C., Caron, M. G., and Lefkowitz, R. J. (1989) P-adrenergrc receptor kinase: primary structure delineates a multigene fam11y.Science 246,235-240 12. Benovic, J. L., Onoranto, J. J., Amza, J. L., Stone, W. C., Lohse, M., Jenkins, N A., Gilbert, D. J., Copeland, N. G., Caron, M. G., and Lefkowitz, R. J. (1991) Cloning, expressron, and chromosomal localization of j3-adrenergrc receptor kmase 2. J Bzol. Chem. 266, 14,939-14,946. 13. Kunapuli, P. and Benovtc, J. L (1993) Cloning and expression of GRKS: a member of the G protein-coupled receptor kinase family. Proc. Natl. Acad. Scl USA 90,5588-5592. 14. Benovic, J. L. and Gomez, J. (1993) Molecular clomng and expression of GRK6. J Biol. Chem 268, 19,521-19,527.

19 Radioligand Binding Measurement of Receptor Sequestration in Intact and Permeabilized Cells Diana M. Slowiejko

and Stephen K. Fisher

1. Introduction A frequently observed adaptive response to chronic agonist occupancy of cell surface receptors is a process in which receptors become internalized or sequestered into a cell compartment to which hydrophilic ligands have only a limited access (I-3). Although receptor sequestration has frequently been documented, its physiological significance and underlying mechanism(s) remain obscure. This is largely the result of the routine use of intact cells for the measurement of sequestration events. Although, in principle, the use of permeabilized cells would appear to provide an ideal means whereby receptor sequestration could be monitored under conditions in which the intracellular milieu is manipulated, there has been little systematic use of this type of preparation. Moreover, previous attempts to monitor the internalization of /3-adrenoceptors in permeabilized cells by means of radioligand binding studies have been unsuccessful (4,s). In this chapter, we describe an agonist-induced sequestration of muscarinic cholinoceptors in intact human SH-SYSY neuroblastoma cells and in preparations of the same cells rendered permeable by the addition of either digitonin, a nonionic detergent, or two bacterial toxins, namely, streptolysin-0 or the a-toxin from Staphylococcus aureus (6). In these preparations, a selective loss of muscarinic receptor sites that are labeled by the hydrophilic radioligand [3H]N-methylscopolamine (NMS) is observed in response to agonist addition, but the total number of muscarinic receptors remains unchanged. The characteristics of receptor sequestration in permeabilized cells resemble those of intact cells (7,s). From

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2. Materials 2.1. Measurement intact Cells

of Muscarinic

Receptor Sequestration:

1. Assay and working buffer (buffer A): 142 mMNaC1,5.6 mMKCL2.2 mMCaC12, 3 6 mMNaHCOs, 1 nnI4 MgC12, 5.6 mM n-glucose, and 30 nnI4 HEPES-NaOH buffer, in detomzed water, pH 7.4. Buffer A solution may be stored at 4°C and n-glucose (1 mg/mL) added just prior to use. 2. Stock solutions of muscarmic agomst and antagonist: 10 nuI4 oxotremorine-M (Research Brochemicals, Natick, MA) and 1 nnI4 atropine sulfate (Sigma, St. Lotus, MO) dissolved m buffer A. Both stock soluttons may be stored for at least 2 mo at -20°C. 3. Radiolabeled muscarinic antagonists: [3H]N-methylscopolamine ([3H]NMS, a quaternary N+ hydrophrlic antagomst) and [3H]qumuclidinyl benzrlate (C3H]QNB, a tertiary N lipophrlrc antagonist). (Both radioligands are obtained from New England Nuclear, Boston, MA.) Stock reagents are obtained in ethanol and stored at -20°C. Prror to assay, stock solutions of antagomsts are diluted to the desired final concentration in appropriate buffer (radioligand buffer solution; see Note 1). 4. Binding reaction tubes: 5-mL polypropylene tubes in racks that can be accommodated in a Brandel cell harvester (Brandel, Gaithersburg, MD). 5. Vacuum filtration: Brandel cell harvester, 0.9% (w/v) NaCl in deionized water, Whatman GF/B glass fiber filters, small forceps, 20-mL scintillation vials, and Umversol scintillation fluid.

2.2. Measurement of Muscarinic Permeabilized Cells

Receptor

Sequestration:

1. Assay and working buffer (KGEH): 139 mit4potassmm glutamate, 2 rmI4 ATP, 4 mM MgCl,, 10 mM EGTA, 2 2 mA4 CaCl, (free [Ca2’] -60 nM; see Note 2), and 30 nnI4 HEPES-NaOH buffer, pH 7.4. With the exception of ATP, 10X concentrated stock solutions, prepared in deionized water, may be stored at -20°C. ATP solutrons should be prepared just prror to use. Stock EGTA solutions must first be dissolved in a minimal volume of 2MNaOH, then diluted with deionized H,O. 2. Stock solutions of 10 mM oxotremorme-M and 1 mM atropine sulfate dissolved in KGEH. Both solutions may be stored for at least 2 mo at -20°C. 3. Permeabilizing agents: Digitomn (Calbiochem, La Jolla, CA) -80% pure. A 2 mM (100X) stock solution in 100% EtOH can be stored at -20°C indefinitely. Stock solutron should be sonicated to dissolve solute. Streptolysin-0 (SLO) (Wellcome Diagnostics) is prepared daily in KGEH buffer at a 10X concentration (= 5 IU/mL). a-Toxin from S aureus (Calbiochem) is prepared dally in KGEH buffer at a 10X concentration (= 2000 U/mL). 4. Assessment of permeabilization efficiency: O&0.5% trypan blue dye stock (w/v m deionized water), hemocytometer, microscope

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3. Methods

3.1. Assay of Receptor Sequestration in Intact Cells 1. Preparation of assay binding tubes: Binding tubes (triplicate replicates for each experimental condition) are placed in a rack in an ice bath. To each tube is added either 50 l.rL of buffer A or atropine (final concentration = 25 w for determination of nonspecific antagonist binding). To these tubes is then added 1.75 mL of radioligand buffer (buffer A/[3H]QNB or buffer AJ3H]NMS; see Note 1). 2. SH-SYSY neuroblastoma cells (passage 70-90, 10-14 d in culture) grown in tissue culture flasks (75 cm*/150 mL) are first detached from the substratum by incubation in a modified Puck’s D, solution (91, centrifuged at lOO-300g for l-2 min at room temperature and then washed once in buffer A (10 ml/flask). The cell pellet is then resuspended in buffer A and divided into two ahquots (10-20 mL) in 50-mL conical reaction tubes (e.g., A = agonist treatment; B = buffer control). The cell suspension is then centrifuged at -lOO-300g for l-2 min at room temperature and buffer A aspirated. Each cell pellet is then resuspended in 4.5 mL of buffer A, and 0 5 mL of either the stock solution of oxotremorine-M (final concentration = 1 mA4), or buffer A, added Incubations are allowed to proceed at 37°C for the desired time (e.g., 30 mm, see Note 3). 3. The reactions are terminated by the addition of 6 vol of ice-cold buffer A (30 mL) to each conical tube, which are then maintained on ice. Following centrifugation at 300g for l-2 min at 4°C and aspiration of the supernatant, the cell pellets are then resuspended in 10 mL of buffer A and recentrifuged. Supernatants are then aspirated and each pellet resuspended in 2 mL of ice-cold buffer A (see Note 4). 4. Aliquots of cell suspension (200 pL) are added to bmding tubes (maintained on ice) prepared as described in step 1. To assure uniform sampling, the cell suspension is continuously stirred, using a magnetic stir bar while 200~@, vol are aliquoted into binding tubes The total volume of the assay is 2 mL (1.75 mL radioligand buffer, 50 pL buffer or atropine, and 200 pL of cell suspension; see Note 5). Tubes are then vortexed to ensure complete mixing and triplicate replicates of each experimental condition are assayed. For example, in an experiment with one agonist treatment and its buffer control, six 200~pL aliquots from each cell suspension will be used for the radioligand binding assay (three tubes with either 50 pL of buffer A or 50 pL of atropine added). The remainder of the sample is retained for determination of protein concentration 5 Binding reactions are then allowed to proceed to equilibrium: For detection of cell-surface binding sites, a saturating concentration of the hydrophilic antagonist [3H]NMS (typically w10 nM final concentration) is used and tubes allowed to incubate for 1g-24 h at 4°C (to prevent receptor recycling). For determmation of the total number of muscarmic receptor sites, a saturating concentration of E3H]QNB (1 .O nA4) is used. Binding reactions are allowed to proceed for 60 min at 37°C. Prior to vacuum filtration, all tubes’ are vortexed. 6. Binding reactions are then rapidly terminated by means of vacuum filtration (Brandel cell harvester) through Whatman GF/B filters, and each filter is washed

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three times with -3 mL of 0.9% NaCl. To each filter sample is then added 5 mL of scintillation fluid and radioactivity quantitated by scintillation counting after a 24 h delay (see Note 6). 7. To ensure that the loss of [3H]NMS binding observed following agonist treatment is reflective of a loss of only cell-surface sites, total receptor number should also be determined by means of [3H]QNB binding. If, under the chosen conditions, a loss of [3H]QNB-binding sites is also observed (i.e., receptor down-regulation has occurred), then this value is subtracted from that obtained for the loss of E3H]NMS sites. For example, if a 50% loss of [3H]NMS sites occurs in the presence of the agonist, and, in addition, a 10% reduction in the number of [3H]QNB sites is observed, then the percent sequestration is calculated to be 40%.

3.2. Assay of Receptor Sequestration

in Permeabilized

Cells

1. Cell permeabilization: buffer (prea. Digitonin: Cell pellets are resuspended in 20 wdigitonin/KGEH pared just prior to use) at a protein concentration of -2-3 mg/mL (see Note 7). Incubate for 5 min at 37°C. b. Streptolysin-0: Cell pellets are resuspended in KGEH buffer that contains Streptolysin-0 at a concentration of 0.5 IU/mL and incubated for 10 min at 37°C. c. a-Toxin: Cell pellets are resuspended in KGEH buffer that contains a-toxin at a concentration of 200 IU/mL and incubated for 30 min at 37°C (see Note 8). 2. Efficiency of permeabilization is assessed by means of trypan blue exclusion (see Note 9). 3. Permeabilized cells are then centrifuged at -2OO-300g for 2-3 min to obtain a stable pellet (see Note 10) and washed with an equal volume of KGEH buffer (minus permeabilizing agent). Cell pellets are then centrifuged again at -300g for 2-3 min and resuspended in KGEH buffer. 4. The permeabilized cell suspension is then divided into 4.5-mL aliquots and 0.5 mL of either agonist stock or KGEH buffer (control) added. 5. Assay procedure. Follow steps described previously for intact cells (Section 3.1., steps 2-7), with the exception that KGEH buffer should be used instead of buffer A.

3.3. Critique The use of permeabilized SH-SYSY neuroblastoma cells offers a simple and convenient approach to monitor receptor sequestration events and their regulation. When performed under controlled conditions, muscarinic receptor sequestration is reliably detected and shares many of the characteristics of those observed in intact cells (e.g., kinetics, agonist efficacy, and potency profiles). However, it should be noted that the absolute degree of receptor sequestration is reduced in permeabilized cells by 30-40% when compared to that observed for intact cells. This may reflect either the loss of necessary cytosolic components or, alternatively, damage to the internalization mechanism. Nonetheless, permeabilized cells provide an opportunity to study the interrelationships

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between receptor activation events and the internalization of receptor. For example, by the use of permeabilized cells, it has been demonstrated that the production of phosphoinositide-derived second messenger molecules is not a prerequisite for receptor sequestration (8). 4. Notes 1. Saturating concentrations of radiolabeled antagonist should be used (i.e., those necessary for occupancy of >95% of receptor sites). This should be determined empirically for each receptor type and cell system utilized. 2. The free [Ca2+] may be manipulated to values other than 60 nM by varying the Ca2+/EGTA ratio (see ref. 10). This value was chosen because cytoplasmic Ca2+ in quiescent neuroblastoma cells approximates this concentration. However, this value should be determined for each cell system studied. 3. Initially, it may be useful to determine the time-course of receptor sequestration in a specific cell system (see Fig. 1 for the kinetics of muscarinic cholinergic receptor internalization in human SH-SYSY neuroblastoma cells). The maximum effect is most frequently observed after 30-60 min of agonist incubation. Furthermore, longer periods of agonist exposure (Le., hours) may result in a loss of

receptorsfrom the cell becauseof downregulation. 4. It is very important that all steps following the termination of the sequestration reaction with ice-cold buffer also be performed at the same low temperature, to prevent recycling or the recovery of the sequestered receptors back to the plasma membrane. Both stages of receptor cycling are inhibited at temperatures below 10°C (see ref. II). Also, it should be noted that a single wash is effective in removing >99% of the hydrophilic agonist, oxotremorine-M (as determined by means of the removal of a tracer dose of [3H]oxotremorine-M added to these tubes). However, more lipophilic agonists will not be removed so readily. Because residual agonist may displace the radioligand and, accordingly, alter the interpretation of the data, this issue needs to be carefully considered. 5. Atropine (25 @4 final concentration) is a muscarinic antagonist used to determine nonspecific radioligand binding to the cells (i.e., by definition, radioligand bound that is not displaced by atropine). This value is subtracted from the total binding values obtained in the absence of atropine to obtain the specific binding values. For example, if the average of the triplicate replicates obtained for total binding is 15,000 dpm, and the corresponding value for cells incubated in the presence of atropine is 1000 dpm, then the specific binding is 14,000 dpm (15,000 - 1000). Ideally, nonspecific binding should be less than 10% of total binding. 6. Data are typically expressed as specific radioactivity bound (dpm/mg protein or fmol of radioligand bound/mg protein). The extent of receptor sequestration that occurs in response to agonist addition is calculated as follows:

% sequestration= 1-

1

[3H]NMS specifically bound in agonist-treated cells x loo [3H]NMS specifically bound in buffer-treated cells

Slowiejko and Fisher

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g .IO=

.-rg L mg

loo-

90-

g: ao‘3 8 Q)%

70-

Time (min) Fig. 1. Time-course of agonist-induced loss of [3H]NMS and [3H]QNB binding sites from digitonin-permeabilized SH-SYSY neuroblastoma cells. Reprinted with permission from ref. 8.

7. The ratio of digitonin:cell protein is critical in selectively permeabilizing the plasma membrane without unmasking sequestered, intracellular ligand binding sites (see Fig. 2). If the concentration of digitonin used is too high, then hydrophilic radioligands may gain access to sequestered sites. The concentration of digitonin chosen should be determined empirically as the lowest concentration of permeabilizing agent required to permeabilize >80% of cells. 8. a-Toxin permeabilization results in the formation of smaller pores than either digitonin or streptolysin-0 and allows for the passage of molecules of -1000 Dalton (see ref. 6). a-Toxin is very selective for the plasma membrane even at higher concentrations (unlike the cholesterol-targeting agent, digitonin). Streptolysin-0 forms pores of comparable size to those obtained for digitonin, and are generally restricted to the plasma membrane. 9. To determine permeabilization efficiency, a stock solution of trypan blue (OA0.5% w/v) solution is diluted lo-fold in KGEH buffer (i.e., final concentration of 0.04%) and then incubated 1: 1 (v/v) with a cell suspension sample for not more than 5 min at room temperature. Make the required dilutions of cell suspension in KGEH buffer to maintain isotonicity. The number of cells that are permeabilized (i.e., those that do not exclude trypan blue) are counted using a hemocytometer. 10. Permeabilization with the detergent, digitonin, often results in an unstable or fluffy cell pellet. Make certain that centrifugation conditions are optimal for obtaining a stable pellet and increase centrifugal force if necessary.

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Binding Measurement [3H]NMS

249

Q

[3H]QNB

60 t

I

0

1

20

40

60

80

[Digitonin]

I

0

20

40

60

80

(PM)

Fig. 2. Sequestered muscarinic receptor sites that are inaccessible to E3H]NMS can be exposed by the use of higher concentrations of digitonin. In this experimental paradigm, intact cells were first incubated for 30 min at 37°C in the presence (solid symbols) or absence (open symbols) of the muscarinic agonist, oxotremorine-M, and then permeabilized for 5 min with digitonin at the concentrations indicated. After the cells had been washed free of detergent, aliquots of cells were taken for either [3H]NMS or [3H]QNB binding. Note that as the digitonin concentration increases, the ability of [3H]NMS to detect sequestered receptors is also enhanced. In contrast, no changes in total receptor number (as monitored by [‘H]QNB) occur under these conditions. Concentrations of digitonin above 80 pA4 result in a loss of muscarinic receptors. Data reprinted with permission from ref. 8.

References 1. von Zastrow, M. and Kobilka, B. K. (1992) Ligand-regulated internalization and recycling of human B,-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J. Biol. Chem. 261,3530-3538. 2. Harden, T. K., Petch, L. A., Traynelis, S. F., and Waldo, G. L (1985) Agonistinduced alteration in the membrane form of muscarinic cholinergic receptors. J. Biol. Chem. 260, 13,060-13,066. 3. Feigenbaum, P. and El-Fakahany, E. E. ( 1985) Regulation of muscarinic cholinergic receptor density in neuroblastoma cells by brief exposure to agonist: possible involvement in desensitization of receptor function. J. Pharmacol. Exp. 7’her.233,134-140. 4. Fratelli, M., Gagliardini, V., and De Blasi, A. (1989) Low affinity of /3-adrenergic receptors for agonists on intact cells is not due to receptor sequestration. Biochim. Biophys. Acta 1012, 178-183.

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5. Lohse, M. J., Benovic, J. L., Caron, M. G., and Lefkowitz, R. J. (1990) Multiple pathways of rapid Pz-adrenergic receptor desensitization. J. Biol. Chem 265, 3203-3209.

6. Ahnert-Hilger, G., Mach, W., F&r, K. J., and Gratzl, M. (1989) Poration by o-toxin and streptolysin 0: an approach to analyze intracellular processes. Methods Cell Biol. 31,63-90. 7. Thompson, A. K. and Fisher, S. K (1990) Relationship between agomst-induced muscarmic receptor loss and desensitization of stimulated phosphoinositide tumover in two neuroblastomas: methodological considerations. J. Pharmacol. Exp. Ther. 252,744-752.

8. Slowiejko, D. M., Levey, A. I., and Fisher, S. K. (1994) Sequestration of muscarinic cholinergic receptors in permeabilized neuroblastoma cells. J. Neurochem. 62,1795-l 803. 9. Fisher, S K. and Snider, R. M. (1987) Differential receptor occupancy requirements for muscarinic cholinergic stimulation of inositol lipid hydrolysis in brain and in neuroblastomas. Mol. Pharmacol. 32,8 l-90. 10. Fisher, S. K., Domask, L. M., and Roland, R. M. (1989) Muscarinic receptor regulation of cytoplasmic Ca2+ concentrations in human SK-N-SH neuroblastoma cells: Ca2+ requirements for phospholipase C activation. Mol. Pharmacol 35, 195-204. 11, Thompson, A. K. and Fisher, S. K. (1991) Preferential coupling of cell surface muscarinic receptors to phosphomositide hydrolysis in human neuroblastoma cells. J. Biol. Chem. 266, 5004-5010.

20 Regulation of Receptor Expression Analysis of Receptor mRNA and Gene Transcription Philip A. Iredale, Maura E. Charlton, and Ronald S. Duman

John D. Alvaro,

1. Introduction In addition to desensitization and downregulation of receptors at the cell membrane, the number of receptors expressed is modulated by regulation of steady-state levels of mRNA and the rate of gene transcription (see refs. I and 2 for reviews). Regulation of receptor mRNA and gene transcription has been observed for many different types of receptors, including those for hormones, neurotransmitters, and neuropepttdes, and may occur in response to exposure to receptor agonists, different types of hormones, or drug treatments (3-9). Thus, regulation of receptor mRNA and gene transcription could represent a mechanism for long-term modulation of receptor expression. The levels of receptor mRNA and gene transcription rate are regulated by complex, intracellular signal transduction pathways. These pathways can lead to regulation of mRNA by two general mechanisms. First, the level of receptor mRNA can be modulated by regulation of its stability. The mechanisms that control mRNA stability appear to involve the presence of RNA-binding proteins that influence mRNA degradation (10-12). These proteins recognize and bind to specific RNA sequences that are usually localized in the 3’ noncoding portion of mRNA. Second, the level of receptor mRNA is dependent on the rate of mRNA synthesis, or rate of gene transcription. Gene transcription is regulated by factors that bind to specific DNA sequences that are usually, but not always, localized in the promoter region of the gene (13,14). These gene transcription factors can be regulated by phosphorylation (e.g., CAMP response element-binding protein) or by regulation of the total amount of transcription From

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factor (e.g., the c-Fos immediate early gene transcription factor). In addition, it is clear that many transcription factors are regulated by both phosphorylation and regulation of the total amount of protein. There are several methods available for analysis of receptor mRNA and gene transcription. Steady state levels of receptor mRNA can be determmed by Northern blot or RNase protection analysis. These two procedures provide a determination of the levels of a specific receptor mRNA species in total RNA that has been extracted from cultured cells or tissues. The successand reproducibility of these two techniques are dependent on the quality of the extracted RNA. The RNase protection assay is more sensitive and is preferred for receptors that are expressed at relatively low levels, as is the case for many G protein-coupled receptors. Both of these methods utilize radiolabeled RNA or DNA probes that are specific for the receptor of interest. To determine the stability of mRNA, the half-life can be determined from the rate of mRNA decay in the presence of gene transcription inhibitor. Finally, the rate of gene transcription can be determined by nuclear run-on, a procedure that measures newly transcribed, radiolabeled RNA transcripts in isolated cell nuclei. This chapter describes the detailed protocols required to determine steadystate levels of mRNA and thereby determine if the expression of a particular receptor is regulated at the level of its mRNA. To determine if the changes in levels of receptor mRNA result from regulation of the stability of mRNA or rate of gene expression, mRNA half-life and nuclear run-on analysis is conducted. These studies are conducted in cultured cell lines that express the receptor of interest. These approaches will provide novel information regarding the molecular mechanisms underlying the regulation of receptor mRNA. Moreover, these studieswill provide new areasof researchon the complex mechanisms that underlie the regulation of mRNA stability and gene transcription.

2. Materials 2.1. Northern Blot Technique 2.1.1. RNA Isolation 1. Diethyl pyrocarbonate (DEPC): All solutions that will come into contact with RNA must be treated with 0.1% (v/v) DEPC. The DEPC-treated double-distilled water must be shaken vigorously or stirred, placed in a 37°C incubator overnight with the caps open, and autoclaved. 2. Cesium chloride: 5.7Mcesmm chloride (CsCl) in DEPC-treated water sterilized

through a Nalgene PES0.2~pmfilter. Store the solution at room temperature. 3. Guanidinium thiocyanate (GIT): 4.OM guanidinium thiocyanate, 0. 1M Tris-HCl, pH 7.5, filter through Nalgene PES 0.2 pm and store at 4°C. Add 0.84% 2-mercaptoethanol (BME) immediately pnor to use.

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Ultracentrifuge

and DEPC-treated ultracentrifuge tubes. acetate, pH 6.0, DEPC-treated. 70 and 100% ethanol stored at -20°C. Autoclaved RNase-free pipet tips and microcentrifuge tubes. 8. Dry ice. 9. Kimwipes. 10. Spectrophotometer. 3M ammonium

2.1.2. Riboprobe

Template Preparation

1 2. 3. 4. 5. 6. 7. 8.

DEPC-treated water. Mim-prepped plasmid DNA containing probe of interest. Restriction enzyme that has a blunt end or 5’ overhang restriction site. Protemase K (Boehringer Mannheim [Indianapolis, IN]) 10% SDS m DEPC-treated water Phenol/chloroform/isoamyl alcohol: (50:49: 1) 3M DEPC-treated sodium acetate, pH 6.0. 70 and 100% ethanol stored at -2O’C. 9. Dry ice. 10. 1% agarose gel and DNA electrophoresis apparatus. Il. Autoclaved microcentnfuge tubes.

2.1.3. Riboprobe

Transcription

1. Stratagene RNA transcription kit: 5X transcription buffer, 0.75M dithiothreitol, 10 mMnucleotldes (ATP, GTP, CTP, UTP). Premix three nucleotides, excludmg the nucleotide that will be used to radiolabel the probe. Store all solutions at -20°C. 2. RNase block (Stratagene). 3. T3, T7, and/or SP6 RNA polymerases (Stratagene, La Jolla, CA) 4. 10 U/pL RNase-free DNase (Stratagene). 5. DEPC-treated water. 6. DEPC-treated 0. 1M EDTA. 7. [w~*P]-NTP (800 Ci/mmol, New England Nuclear [Boston, MA]) for Northern blots and RNase protection assay; [cz-~?$or CZ-~~P]-NTPSfor in sztu hybridization. 8. STE: 100 mMNaCl,20 mM Tris-HCl, pH 8.0, 10 mMEDTA. Individual solutions should be DEPC-treated, except Tris, which should be made in DEPCtreated water. 9. NUCTRAP push column (Stratagene). 10. Autoclaved microcentrifuge tubes.

2.1.4. Random Primed Labeling of DNA 1. Maxi (or multiple mini)-prepped plasmid DNA containing full length or fragment of target sequence. 2. Restriction enzymes that cut out fragment of interest with complete removal of vector DNA.

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3. Random primed labeling kit (Gibco [Grand Island, NY] or Boehringer Mannheim), which includes dNTPs, reaction mixture containing hexanucleotide primers, and Klenow fragment. All of these reagents can be purchased separately. 4. [a-32P]-dNTP (3000 Ci/mmol, New England Nuclear). 5. Nick purification column (Pharmacia [Piscataway, NJ]).

2.1.5. RNA Formaldehyde

Gel

1. Agarose Sea Kern LE agarose (I .2%) (FMC Corp [Rockland, ME]) or other suitable RNase free agarose. 2. 3-N-Morpholine-propanesulfonic acid (MOPS) 20X buffer: 400 &MOPS, 100 & sodium acetate, 20 mM EDTA. Because MOPS is light sensmve, it should be stored at room temperature in a bottle wrapped in aluminum foil. 3. Sample buffer: 50% formamide, 1.1X MOPS, 6 6% filtered formaldehyde, 7% glycerol, 0.1% bromophenol blue. Store in aliquots at -2O’C. 4. 20X SSC: 3MNaC1, 0.3MNaCitrate, pH 7.0, with HCI. 5. Ethidium bromide: Generally available in a 10 mg/mL stock 6. Formaldehyde: 37% solution filtered with 0.22~)lm syringe filter, heated to 37°C

2.1.6. Capillary Transfer of RNA to So/id Support 1. Whatman 3MM filter paper. 2. Nitrocellulose: 22 inn Nitropure nitrocellulose from Micron Separation Systems (Fisher Scientific). Other nylon membranes commonly used include Biotrans (ICN [Irvine, CA]) and Zeta-Probe GT (Bio-Rad [Hercules, CA]). 3. Plastic reservoir, open ended plastic stand, plastic block, weights.

2. I. 7. Hybridization 1. Hybridization buffer: This buffer works for either riboprobes or cDNA probes: 50% formamide, 4X SSC, 20 mA4 Tris-HCl, pH 8.3, 1X Denhardt’s solution, 0.1% SDS, 10% Dextran sulfate. Store in 50-mL aliqouts at -20°C. 2. Salmon sperm DNA (Sigma [St. Louis, MO]). 3. Hybridization oven or water bath that can reach 65°C.

2.1.8. Posthybridization 1. 2. 3. 4. 5.

Washes

20x ssc. 10% SDS. Wash containers. Water bath. Cassettes, X-ray film (Hyperfilm,

2.2. The RNase Protection 2.2.1. Probe Preparation

Amersham [Arlington Heights, IL]).

Assay

Refer to discussion of riboprobe

preparation

in Section 2.1.3.

255

Regulation of Receptor Expression 2.2.2. End-Labeling DNA Size Markers

1. Commercially available digested DNA size markers ranging from cl00 bp to >l kb. Recommended are $X174-HaeIII digest markers (New England Biolabs) resuspended at a concentration of 1 pg/& 2. [a-32P]-dNTP (New England Nuclear) suitable for the enzyme used for digest (dGTP for Hue111 markers). 3. Klenow enzyme and 10X reaction buffer (Boehringer Mannheim) 4. 4Mammonium acetate, pH 7.5. 5 70 and 100% ethanol stored at -20°C. 6. Dry ice.

2.2.3. Sample Hybridization Hybridization buffer: 80% formamide, 40 &PIPES, EDTA. Individual solutions should be DEPC-treated DEPC-HZ0 is prepared (see Section 2.5.).

pH 6.4,0.4MNaCl, 1 mM in the same manner that

2.2.4. Denaturing Polyacrylamide Gel Preparation 1. Bio-Rad Mini-PROTEIN II Electrophoresis Cell with two sets of plates, four 0.75mm spacers, and two 1O-well combs. 2. Acrylamide/Bis (19: 1) premixed powder (Bio-Rad) 3. Urea powder (ultrapure, American Bioanalytical matick, MA]). 4. 10% APS: 100 mg APS (Bio-Rad) in 1 mL double-distilled deionized H20. Store at 4’C and replace every 2-3 wk. 5. TEMED (Bio-Rad). 6. 1X TBE: 50 mM Tns base, 50 mM boric acid, 1 mM EDTA. 7. lo-mL syringe and 20-gage needle.

2.2.5. Sample Treatment 1, RNase digestion buffer: 10 mMTris-HCl, pH 7.5,300 mMNaCl,5 rniV EDTA. 2 20 m&r& RNase A and 105 U/mL RNase Tl (Boehringer Mannheim). 3. DEPC-treated H,O. 0.1% (v,v) diethyl pyrocarbonate in double-distilled deronized H,O. Incubate 37°C overnight and autoclave. 4. 20% SDS in DEPC-treated H20. 5. Proteinase K (Boehringer Mannheim) 6. Phenol/chloroform/isoamyl alcohol: 1 vol phenol + 1 vol chloroform/isoamyl alcohol (49: 1). 7. 10 pg/mL tRNA (Sigma). 8. 95 and 100% ethanol. 9 Gel loading buffer: 80% formamide, 1 mM EDTA, 0.1% bromphenol blue, 0.1% xylene cyanol. Bromphenol blue and xylene cyan01 should be made up as 10% solutions in DEPC-treated HzO. 10. Whatman 3MM chromatography paper and plastic wrap. 11, Hyperfilm-MP (Amersham), film cassette, and intensifier screen.

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Half-life

Studies

Actinomycin D (Calbiochem). 2.4. Nuclear Run-On 2.4.1. Preparation of cDNA and Gel Purification 1 2. 3. 4. 5. 6. 7 8. 9. 10. 11. 12. 13. 14.

Plasmtd Maxi-Prep kit (Qiagen [Chatsworth, CA]). Restriction enzymes. Low-melt agarose (Bio-Rad). 50X TAE buffer; (1 L) 242 g Tris-base, 57 mL glacial acetic acid, 100 mL OSM EDTA, pH 7.4. Ethidium bromide. Horizontal gel electrophoresis tank. 6X DNA-loading buffer; 0.25% bromophenol blue, 40% (w/v) sucrose. UV transilluminator. Razor blades. 1.5~mL Eppendorf tubes. TE buffer I: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. TE buffer II. 10 mA4 TrtsHCl, 1 WEDTA, pH 7.4. Dry ice. 3M sodium acetate. Ethanol.

2.4.2. Preparation of cDNA Slot Blot 1. 2 3. 4. 5. 6. 7. 8. 9.

DEPC-treated water. 1MNaOH 1.5~mL Eppendorftubes. 6X SSPE diluted from 20X stock* 3M NaCl, 200 mM NaH2P04, Na2EDTA, pH 7.4. Biorad Bio-Dot SF Microfiltration apparatus. Biotrans nylon membrane (ICN). Colored pencil UV crosslinker. Baking oven.

2.4.3. Prehybridization

20 mM

of the cDNA Slot Blots

1. 50-mL Falcon tubes. 2 Salmon sperm DNA (10 mg/mL; Stgma). 3. 100X Denhardt’s solution: 2% Ficoll400,2% polyvinylpyrrolhdine, 2% bovine serum albumin. 4. Prehybridization solution: 50% formamide, 5X SSPE, 5X Denhardt’s solution, 1% SDS. 5. Rotating hybridization oven.

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2.4.4. Preparation of Nuclei for Nuclear Run-On 1. Buffer I* 10 mM Tris-HCl, pH 8.3, 10 mA4 NaCI, 2.5 miV MgCl*, and 5 mM dithiothreitol (DTT). 2. Triton X-100 (Sigma) 3, Sucrose (Sigma). 4. Phosphate-buffered salme (PBS; Gibco). 5. Dounce homogenizer(s) and Type A pestle(s). 6. Glycerol buffer: 50 mM Tris-HCI, pH 8.3,5 r& MgC12, and 40% glycerol. 7. Hemocytometer. 8. IO-mL Superspeed centrifuge tubes (Sorval [Wilmington, DE]). 9 Sorval Superspeed refrigerated centrifuge with fixed angle rotor.

2.4.5. Nuclear Run-On 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 2 1. 22.

2-mL Eppendorftubes with screwcaps and 0 rings (PGC Scientific [Frederick, MD]). [c+~~P]UTP (NEN). 2X RX buffer: 10 mA4Tris-base, pH 8.3, 5 mA4MgC12, 0.3MKCl. RX mix (for six samples); 1.5 mL 2X RX buffer, 15 pL 100 mA4ATP, CTP, GTP, and 7.5 pL 1MDTT Shaking water bath. RNase-free DNase (1 U/mL; Promega [Madison, WI]). 100 mA4 CaCl, solution. 20X SET mix: 10% SDS, 100 n&fEDTA, 200 mit4Tris-HCl, pH 7.4. PK mix (for 6 samples); 20 pL proteinase K solution (16 mg/mL; Boehringer Mannheim), 200 mL 20X SET mix; 80 PL baker’s yeast tRNA (10 mg/mL; Sigma). 26-gage 0.5 needle, l-n& syringe (B-D). Guanidium thiocyanate solution with P-mercaptoethanol (GIT with BME; see Section 2.1.1.). 2M sodium acetate solution. Saturated phenol solution (Amresco [Solon, OH). Chloroform/isoamylalcohol(49: 1). Bench microfuge. Glycogen (20 mg/mL; Boehringer Mannheim). Propanol. 70% ethanol. Scintillation counter and liquid scintillant. 10% SDS. Saran wrap. Phosphoimager or image analysis system with X-ray film.

3. Methods 3.7. Northern B/of Technique Northern blot analysis identifies a specific mRNA species, using radiolabeled probes that contain the complimentary sequence to that of the mRNA. Total RNA or poly(A+) selected RNA is size-fractionated on an agarose gel

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and then transferred to a solid filter. The filter is then hybridized with the radiolabeled probe and the specific RNA band is visualized by autoradiography. There are some advantages of the Northern blot technique over the RNase protection assay. First, Northern blot is a technically more simple protocol. Second, Northern blot analysis allows you to visualize the full-length mRNA species, to confirm that the probe is hybridizing with the specific mRNA of interest. Even if the majority of the RNA studies will be conducted by RNase protection, it is important to confirm the specificity of the riboprobe by Northem blot analysis. Third, both random prime-labeled DNA probes and riboprobes can be used for Northern blot analysis.

3.1.1. RNA Isolation 1. For preparation of RNA from tissue: The tissue should be removed quickly from the animal and frozen immediately on dry ice (see Note 1). 2. Polytron frozen tissue in 5 mL of GIT in a 50-mL tube (see Note 2), and centrifuge at 500g to reduce foam. Alternatively, incubate tubes on ice until foam dissipates. 3. For preparation of RNA from cultured cells: The media is removed and 4 mL of GIT is added to the culture dish containing -1 x 1O6cells. 4. Carefully transfer GIT/RNA solution to ultracentrimge tubes containing 4 mL cesium chloride. Add GIT until tubes are filled to approx 1 cm from the tube rim 5. Place tubes in ultracentrifuge buckets and balance all tubes to the same weight with GIT 6. Centrifuge samples at 150,OOOg at 22°C for 2 1 h. 7. Aspirate or decant supernatant, and wipe remaining liquid from walls of tubes using Kimwipes (see Note 3). 8 Thoroughly resuspend the RNA pellet in 360 pL DEPC-treated water by repeat pipeting (see Note 4). Transfer RNA to a microcentrifuge tube. 9. Add 40 pL of 3M sodium acetate (RNase-free) to each sample and mix. 10. Add 2.5 vol(lOO0 pL) ice-cold 100% ethanol to samples, vortex, and either place on dry ice for 30-45 min or precipitate overnight at -2OOC. 11 Spin for 15-30 min at 17,000g at 4°C. 12. Remove supernatant and resuspend pellet in 70% ethanol. 13. Spin RNA for 3 min at 17,000g at 4’C. 14. Remove supernatant, air dry RNA, and resuspend in DEPC-treated water. 15. Quantitate RNA at OD,,, (OD,,, l= 40 ng RNA/l pL) (see Notes 5 and 6). 16. Store RNA at -80°C

3.1.2. Riboprobe Template Preparation 1. In an autoclaved microcentrifuge restriction enzyme buffer, 40-60 water to a final volume of 100 pL 2. Incubate in water bath for 3-5 h tion enzyme.

tube, mix 10 pg of plasmid DNA, 10 pL of U of restriction enzyme, and DEPC-treated (see Notes 7 and 8). at temperature recommended for the restric-

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259

3. Add 5 pL 10% SDS and 5 p.L 2 mg/mL Proteinase K and incubate for 45 min at 37°C. 4. Add 140 pL DEPC-treated water and extract twice with 250 pL phenol/chloroform/ isoamylalcohol. 5. Precipitate DNA: To the aqueous phase, add 25 cls, 3M sodium acetate and 700 pL cold 100% ethanol. Mix thoroughly by inversion and precipitate on dry ice for 20 min. 6. Centrifuge for 20 min at 17,OOOg at 4°C. 7. Wash pellet in cold 70% ethanol and centrifuge for 5 min at 17,000g at 4OC. 8. Remove ethanol and lyophilize pellet. 9. Resuspend pellet in 40 pL DEPC-treated water (approximate DNA concentration is now 250 ng/&). 10. Run 2 pL DNA on 1% agarose gel to verify completeness of digestion and approximate percent recovery of DNA. 11. Store DNA template at -20°C.

3.7.3. Riboprobe Transcription 1. Warm to room temperature: 5X transcription buffer, 0.75M dithlothreitol, premixed nucleotides, DNA template, and [c+~~P]-NTP (see Notes 9-l 1). 2. In an autoclaved mlcrocentrifuge tube, add 5 pL transcription buffer, 1 pL dithiothreitol, 3 pL premixed nucleotides, 1 p.L RNase block, 1 pg DNA template, 7 pL [cz-~~P]-NTP, 1 pL undiluted polymerase (see Note 12), and enough DEPC-treated water for a total volume of 25 pL. Make sure to add the polymerase last. 3. Pulse spin the sample in a microcentrifuge to mix and remove bubbles, and then incubate for 1 h at 37°C. 4. Destroy the DNA template by adding 10 U of DNase and incubating for 15 min at 37°C (see Note 13). 5. Inactivate the enzymes by adding 1.3 @-.0.5M DEPC-EDTA. 6. Add DEPC-treated water to a final volume of 70 $ and column puni@ on an STEequilibrated NUCTRAP push column, according to manufacturer’s instructions. 7. Count the eluate to determine counts per minute. 8. Store probe at -2O’C and use within 1 d.

3.1.4. Random Primed Labeling of DNA 1. DNA fragment to be random primed labeled must be gel purified. There are numerous methods and kits to accomplish this, which will not be discussed here (see Notes 15-l 7). The Compass DNA purification kit (American Bioanalytical) is highly recommended. 2. The DNA (50-100 ng) dissolved in distilled water must be denatured at 100°C for 5-10 min, followed by quick-cooling on ice. 3. Reaction mix is added to the tube to final concentrations of 0.2MHEPES, 50 mM Tris-HCl, 5 @4 MgC&,lO mM BME, 0.4 mg/mL BSA, 5.4 ODzao U/mL oligodeoxyribonucleotide hexamer primers, pH 6.8 (Gibco reaction mixture).

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4. The cold nucleotides (except for dNTP chosen for radioactive labeling) are added to a final concentration of 20 fl each. 5. Bring reaction mix up to a volume of 44 pL with distilled water. 6. Add 5 pL. of the [a-32P]-dNTP and mix. 7. Add Klenow fragment (1 pL or 3 U) to the tube. 8. Incubate reaction for 2 h at room temperature. 9 Stop reaction by heating to 65°C for 10 min or by addition of 8 NEDTA. 10. Purify the probe on a Nick column according to the manufacturer’s protocol. Il. Count an aliquot of the probe to determine the amount of incorporated radloactivity.

3. I. 5. RNA Formaldehyde

Gel

1. Preparation of gel (see Notes 18-20): MIcrowave at medium setting 0.9-1.2% agarose in 1X MOPS until the agarose is in solution. When the flask is cool enough to grasp, add 2 pL of ethidium bromide, a final concentration of 2% formaldehyde (6.2 mL of a 37% formaldehyde solution), and double-distilled autoclaved water to a total volume of 115 mL. In a fume hood, pour mixture into casting tray with combs and allow to solidify (>45 mm). 2. Thaw RNA on ice, aliquot into RNase-free tubes, and lyophilize. 3. Resuspend RNA pellets in 10-15 pL of sample buffer, heat to 99°C for 2 mm, flick samples, and heat for another minute. Pulse spin tubes in microfuge. 4. Place gel in electrophoresis tank filled with 1X MOPS, and load the RNA samples mto the gel wells and sample buffer into the unused wells (see Note 2 1). 5. Run the gel at 60-80 V for 4-5 h, or until the bromophenol dye is 3/4 through the gel (see Note 22). 6. Place the gel onto plastic wrap (see Note 23) and photograph on UV transllluminator to visualize the ribosomal RNA (28S, 18S, which correspond to approx 5 and 2 kb, respectively). This will also demonstrate the quality of the RNA (i.e., if degradation has occurred). 7. Wash gel twice for 15 min in 10X SSC, while gently shaking to remove the formaldehyde.

3.1.6. Capillary Transfer of RNA to Solid Support 1. Cut mtrocellulose and 2 Whatman 3MM filters the same size as the gel. Also, cut a wick the same width as the gel, but approx 30 cm long 2. Set up gel transfer apparatus as follows (see Notes 24 and 25): Fill reservoir with 10X SSC; place wick on a platform suspended above reservoir and submerge both ends of wtck in reservoir; place gel on the wick with the open side of the wells facing down; prewet mtrocellulose in 2X SSC and place on top of the gel; prewet 2 Whatman filter papers in 10X SSC and place on top of the mtrocellulose; add 16-20 absorbent filters, 11 x 14 cm (available from Gibco), or a stack of paper towels cut to the appropnate size; place glass/plastic plate on top of transfer apparatus; on top of plate place heavy objects, such as two 250~mL bottles, with approx 100 mL water in each.

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261

3. Transfer for approx 18 h. 4. Dismantle transfer apparatus and view nitrocellulose on transilummator. With a pencil, mark the orientation of gel and location of ribosomal bands on the side of the nitrocellulose. 5. UV crosslink the nitrocellulose or bake it at 80°C for 2 h. 6. Store blot m plastic wrap in the refrigerator or use immediately.

3.1.7. Hybridization 1. Immediately prior to hybridization, denature salmon sperm DNA for 10 min at 100°C and quick-cool on ice. Add DNA to the buffer at a final concentration of 100-200 pg/mL. 2. For hybridization in water bath: Place blot in a heat sealable pouch (16.5 x 20.3 cm) with 10-15 mL of hybridization buffer and denatured salmon sperm DNA. Carefully seal and place in shaking preheated water bath. 3, For hybridization in oven (see Notes 26 and 27): Place blot between two pieces of mesh and roll up from bottom to top. Place mesh/blot in hybridization tube and add 15-20 mL of hybridization buffer. Weigh and balance tubes and place in preheated rotating oven. 4. Prehybridization: l-2 h at 42’C (cDNA) or 65°C (riboprobe). 5. Denaturation of probe: Heat riboprobes at 85°C for 5 min. Heat cDNA probes at 95-100°C for 5 min. Both should immediately be quick-cooled on ice before use. 6. Hybridization: Add probes to hybridization buffer with or without additional buffer (see Notes 28 and 29) Hybridization reaction should proceed overnight

3.1.8. Posthybridization

Washes

1. If washes are in hybridization oven, then pipet buffer out of the tube and dispose in liquid radioactive waste. Add 2X SSC, 0.1% SDS heated to hybridization temperature (see Note 30) to the tube and place back in the rotating oven for 20 min. 2. If hybridization was performed in a sealable pouch, carefully remove the blot from the pouch and place in a container for washing. Discard remainder of the buffer in liquid radioactive waste. Wash blot in several hundred milliliters of 2X SSC, 0.1% SDS at the hybridization temperature 3. Additional washes (see Note 3 1): The remaining washes are in increasingly stringent conditions in shaking water baths Riboprobe hybridized blots are washed successively in 2X SSC, 0.1% SDS at 65”C, 0.5X SSC, 0.1% at 65’C, and 0.1X SSC, 0.1% SDS at 68°C. Blots hybridized with cDNA probes are washed in 2X SSC, 0.1% SDS at 55’C, 0.5X SSC, 0.1% SDS 55°C and 0.1X SSC, 0.1% SDS 58°C. Wash at each condition for 20-30 min and check radioactivity remaining on blots after each wash, with a hand-held counter. 4. Cover blot in plastic wrap and expose to film. Store blot in plastic wrap for hybridization with additional probes. 5. The method of quantitation depends on the equipment available in the laboratory. Phosphoimagers work well for RPA and Northern blot quantitation and produce publishable quality images. Exposure to film is also important after analysis

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to preserve a hard copy of the image. Another possibility is to quantitate an autoradiogram using an image-analysis system and the National Institutes of Health image analysis program. Normalize data with the internal standard.

3.2. The RNase Protection Assay The RNase protection assayutilizes a radiolabeled rlboprobe that is complimentary to the RNA of interest. Hybridization of the riboprobe with total RNA is conducted in solution, and the sample is subsequently treated with RNase to

degrade unhybridized probe. Only the specific RNA:RNA hybrids are resistant to RNase treatment, so that nearly all nonspecific background is removed. The protected RNA hybrids are then visualized by autoradiography. There are several advantages of the RNase protection assay. Most importantly, it is more sensitive than the Northern blot and therefore allows for analysis of rare mRNA

species. The other major advantage 1sthat it 1svery specific and there is very low background because of the posthybridization RNase treatment. However, the RNase protection assay 1smore technically difficult. A very specific probe must be used, which means that in most cases the sequence of the riboprobe must be derived from the same species as that of the RNA to be analyzed. If the sequence of the riboprobe is not identical, the mismatched regions of the probe will be susceptible

to RNase digestion.

You should also verify that the

sequence that you have chosen is specific for the receptor of interest. For example, the transmembrane regions of many receptors within a family can be very similar. The riboprobe should also be of an appropriate length (i.e., -lOO500 bp), as larger probes are susceptible to breakdown and will yield multiple bands on a gel. 3.2.1. Probe Preparation 1. Label and purify receptor-specific riboprobe according to instructions in Section 3.1.3. Use only riboprobes with a percent incorporation greater than 30% (see Notes 32 and 33). 2. Dilute an aliquot of probe to 1 x lo5 counts/min/& m DEPC-Hz0 and store at 4°C. The remainder of the probe should be stored at -20°C.

3.2.2. End-Labeling

DNA Size Markers

1. In a total volume of 20 $, mix 1 a DNA size markers and 2 & 1OX Klenow &zyme buffer (see Note 34). 2 Heat DNA for 3 min at 8O’C. 3. Add 10 @i of appropriate [a-32P]-dNTP and 1 U Klenow enzyme. 4. Incubate for 10 mm at room temperature. 5. Precipitate the labeled DNA by adding an equal volume of 4M ammonium acetate and to the new volume, 2.5 vol of Ice-cold 100% ethanol Place on dry Ice for 20 min.

Regulation of Receptor Expression 6. 7. 8. 9. 10

263

Centrifuge at 17,000g for 20 mm at 4’C. Rinse pellet in cold 70% ethanol and lyophilize. Resuspend in 50 pL double-distilled deionized H,O. Count 2 & and dilute small aliquot to 1000 counts/min/pL. Store diluted and undiluted aliquots at -20°C. Markers are usable for several weeks.

3.2.3. Sample Hybridization 1. Lyophilize RNA samples in 1.5~mL autoclaved microfuge tubes and resuspend completely in 29 $ hybridization buffer. 2 Add 1 pL of diluted probe to each sample and heat for 10 mm at 85°C. 3. Quickly transfer the samples to a 65°C water bath and hybridize 16-18 h

3.2.4. Denaturing Polyacrylamide Gel Preparation 1, For two 8% polyacrylamide mini-gels, add 1.2 g acrylamidelbu (19: 1) and 7.2 g urea (final concentration of 8M) to 15 mL 1X TBE m a 50-mL Falcon tube (see Notes 35 and 36). 2. Shake the tube and heat at 65°C for at least 15 min to dissolve solutes completely. 3. Cool the solution to approximately room temperature and filter through Whatman No. 1 filter paper into another 50-mL Falcon tube. 4. Add 90 pL 10% APS and 9 pL TEMED to the tube and mix thoroughly without creating air bubbles. 5. Using a lo-mL syringe, pour the gel solution into each mini-gel casting apparatus. 6. Insert IO-well combs and allow gels to polymerize >2 h. 7. Just prior to use, run each apparatus under cold tap water to remove excess acrylamide from combs. 8. Place gels in buffer chamber and fill with 1X TBE accordmg to manufacturer’s instructions. 9. Within l-3 min of sample loading, clear urea from each well using 1X TBE in a lo-mL syringe with a 20-gage needle.

3.2.5. Sample Treatment 1. To each hybridized sample, add 400 $ RNase digestion buffer with 25 pg/mL RNase A and 125 U/mL RNase Tl . 2. Mix thoroughly by inversion and incubate 45 min at 37OC. 3. Add 20 @., 10% SDS and 50 pg Proteinase K and mix by inverting 5-7 times. Incubate for 15 min at 37OC. 4. Add 400 pL phenol/chloroform/isoamyl alcohol, vortex 10 s, and spin in microfuge for 1 min. 5. Transfer aqueous phase to new 1.5-mL tube and precipitate RNA as follows: add 1 $ 10 mg/mL tRNA carrier and 1 mL ice cold 100% ethanol and mix vigorously by inversion. Place on dry ice for 20 min. 6. Spin samples in microfuge for 20 min at 17,000g at 4’C. 7. Wash pellets with 95% ethanol and spm m microfuge for 5 min at 4°C.

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8. Remove ethanol and lyophilize samples. 9. Thoroughly resuspend pellets in 10 pL gel loading buffer. 10. If radiolabeled DNA size markers are to be used, 1000 counts/mm of 32P-dNTP end-labeled DNA should be added to 10 $ loading buffer. 11. Incubate all samples for 5 min at 95OC, pulse spin in microfnge, and quack-cool on ice 12. Load samples onto gels, and run at 200 V until xylene cyan01 dye front has run to the bottom of the gels (see Notes 37-40) 13. Fix gels in 15% methanol/5% acetic acid for 10 mm. 14. Transfer gels onto Whatman 3 MM Chromatography paper and cover with plastic wrap. 15. Dry gels for 40 min at 8O’C. 16. Expose to X-ray film with an intenstfier screen overnight.

3.3. Receptor Half-Life Studies The half-life of mRNA in cultured cells can be determined by measuring the decay of the mRNA in the presence of a DNA transcription inhibitor. The mRNA can be measured by either Northern blot or RNase protection analysis. 1. Pretreat cells with agonist of interest for time period which shows maximum effect. 2. Add actinomycin D (2 mg/mL) to these cells as well as to control cells, harvest at 0.5, 1,2, 3, and 4 h after addition of the transcription inhibitor (see Note 41). 3 Perform RNase protection assays (see Sectton 3.2 ) on RNA isolated (see Section 2.2.1.) from these samples, as well as from control and agonist-treated cells not treated with actinomycm D. 4. Compare the rate of decay of the mRNA species of interest in control and agomst-treated cells (see Note 42)

3.4. N&ear

Run-On

The nuclear run-on assay provides a measure of the rate of gene transcription. Cell nuclei are isolated from cultured cells andthen incubated with [cI-~~P]T.-JTP and unlabeled NTPs in order to label nascent RNA transcripts. [a-32P]-labeled RNA IS then purified and specific RNA transcripts are detected by hybridization to cDNA that is immobilized on nylon or nitrocellulose membranes. The level of hybridization to the cDNA IS a measure of the transcription rate. This technique provides important information on the regulation of transcription rate, but it is technically

very demanding.

(Don’t

get discouraged

if you don’t

succeed the first time; it usually takes several attempts to obtain useful results.) 3.4.1. Preparation of CDNA and Gel Purification 1. Isolate at least 500 ng of the plasmid the Qiagen Maxi Prep ktt

containing the cDNA(s) of interest, using

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2. Incubate plasmid overnight at 37°C with the appropriate restriction enzymes to excise the cDNA of interest from the vector. 3. Pour a 100~mL low-melt agarose gel containing 0.8, 1, or 1.5% agarose (dependent on the size of cDNA of interest), 0.01% (w/v) ethidium bromide in TAE. 4. Add DNA-loading buffer to the cut plasmid and load the low-melt agarose gel (see Note 43). 5. Electrophorese the gel at approx 50-70 mV until there is complete separation of the cDNA of interest from the vector. 6. Locate the bands of interest with a UV transilluminator and use a sharp razor blade to cut them from the gel. Trim away as much excess agarose as possible. 7. Place each slice mto a 1.5~mL Eppendorf tube and add 3 vol of TE buffer, pH 8.0. 8. Incubate at 70°C for 5 min to melt the agarose, and mix gently. 9. Quick-freeze the samples m dry ice for 30 min. 10. Thaw the samples, tap gently, and centrifuge at 17,OOOg for 5-10 min. 11. Remove the supernatant and ethanol precipitate the DNA by addition of l/l0 vol of 3M sodium acetate and 2 vol of ethanol. 12. Resuspend and combine the samples in -100 pL of TE buffer, pH 7.4.

3.4.2. Preparation of CDNA Slot Blot 1. Dilute the cDNA of interest to a final volume of 660 @, of TE buffer, pH 7.4 (for six samples), in a 1.5-n& Eppendorf tube. 2. Add 73.5 & of 1MNaOH. 3. Heat for 10 min at 95°C and place on ice. 4. Add 7.4 pL of 6X SSPE and mix well. 5. Assemble a Bio-Rad Bio-Dot SF Microfiltration apparatus with six pieces of tilter paper (prewetted with 6X SSPE), with one piece of Biotrans nylon membrane on top (see Note 44). 6. Attach the apparatus to a vacuum and apply -300 pL of 6X SSPE through each slot. 7. Apply 120 pL of cDNA to each slot (see Note 45). It is necessary to use at least 5 pg of each cDNA. 8. Mark the locations of those slots containing DNA (see Note 46). 9. Remove the nylon membrane and immediately UV crosslink. 10. Air dry the blot overnight; the following morning bake for 2 h at 80°C. 11. Cut the membrane into strips, each containing the cDNA of interest and the control(s) cDNA (see Note 46). The blots are now ready for prehybridtzation.

3.4.3. Prehybridization

of the CD/VA Slot Blots

1. Denature 1.2 mL of salmon sperm DNA (10 mg/mL) for 10 min at 105’C. 2. Add 1 mL of salmon sperm DNA to 49 mL of prehybridization solution and mix well. 3. Place each blot into a separate 50 mL screw-capped Falcon tube and add 5 mL of prehybridization solution. 4. Prehybridize at 42°C for at least 4 h in a rotating hybridization oven. The blots are now ready for hybridization with the newly transcribed RNA from Section 3.4.5.

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3.4.4. Preparation of Nuclei for Nuclear Run-On It is better to prepare fresh nuclei on the day of the experiment than to rely on

frozen samples. Fresh nuclei can be generated as described by Klely et al. (s). 1. Wash the cell monolayers twice with ice-cold PBS. 2 Add 1 mL of buffer I (see Note 47) to each loo-mm dish and keep on ice for 10 min. 3. Add a further 1 mL of buffer I containing 0 6il4 sucrose and 0.6% Triton X-100 and scrape the cells mto a chilled Dounce homogenizer (see Note 48). 4 Homogenize by six strokes with a type A pestle and layer the homogenate over 2 mL of 0.6M sucrose in buffer I in a 10-mL centrifuge tube (see Note 49). 5 Centrifuge the tubes at 2000g for 10 min at 4°C in a fixed-angle rotor. 6. Rapidly pour off the supematant by invertmg the tube, resuspend the pellet in 200 pL of glycerol buffer, and store on ice (see Note 50) 7. Estimate the number of nuclei using a hemocytometer.

3.4.5. Nuclear Run-On 1. Transfer the nuclei (at least 5 x 10’) to a 2-mL Eppendorf tube (see Note 5 l), add 25 & (250 @i) of [cz-~~P]UTP followed by 200 & of RX mix, into a 2 mL Eppendorf tube, and incubate for 30 min in a 30°C shaking water bath. 2. Add 20 pL of DNase 1 (RNase free; 1 U/mL), followed by 4.6 pL of 100 mM CaCl, solution, and incubate for a further 10 mm at 30°C in the shaking water bath. 3. Add 37 & of PK mix and incubate for 10 min at 37°C in the shaking water bath. 4. Shear the nuclei through a 26-gage 0.5 needle and split into 2 Eppendorf tubes (2 mL) (see Note 52). 5. To each tube add 450 cls, of GIT solution (with BME), 70 & of 2A4 sodium acetate, 800 pL of saturated phenol solution, and 155 & of chloroform/ isoamylalcohol(49: l), mix well, and incubate on ice for 15 min. 6 Centrifuge for 15 min at 17,OOOgat room temperature. 7. Caretilly transfer the aqueous layer to another 2-mL Eppendorftube and add 2 & of tRNA (10 mg/mL), 2 pL of glycogen (20 mg/mL), and an equal volume of propanol 8. Mix well and centrifuge for 15 min at 17,OOOgat room temperature, 9. Remove the supematant, being careful not to disturb the pellet, and wash with 70% ethanol. 10. Air dry or speed-vacuum dry the pellet, resuspend m 100 #. of DEPC-treated water, and recombine the two pellets from each sample. 11. Count 5 pL in duplicate from each sample. 12. Heat the samples to 95°C for 5 min and place on ice. 13. Add an equal number of radioactive counts to each of the prehybrid blots from Section 3.4.3. (see Note 53). 14. Hybridize for 48 h at 42’C in a rotating hybridization oven. 15. Wash the blots m 0.2% SSPE, 0.1% SDS for 10 min at 42°C m a shaking water bath. 16. Wash the blots m 0.1 % SSPE, 0.1 % SDS for up to 10 min at 42’C in a shaking

water bath (seeNote 54).

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17. Dry the blots on filter paper at 37°C for approx 5 min. 18. Cover the blots with Saran wrap and expose to film or a Phosphoimager. 19. Quantitate using image analysis or phosphoimage analysis and normalize the signal from the cDNA of interest with the control cDNA.

4. Notes 1. In order not to overload the GITKsCl density gradient, a maxlmum of 1 g of brain tissue or l/2 g of peripheral tissue per ultracentrifuge tube should be used. 2. The tissue must be homogenized as quickly as possible to avoid degradation of RNA. This can be achieved by freezing the tissue m small pieces (-50 mg), or by rapidly pulverizmg larger frozen tissue sections with a prechilled mortar and pestle. 3. Following CsCl centrifugation, the RNA pellet at the bottom of the tube will be both transparent and gelatinous. 4. The volume m which the final RNA pellet is resuspended depends on amount of RNA expected. It is better to make concentrated samples that can be diluted following quantitation than to make samples that are too dilute to be useful. These latter samples would have to be lyophilized or reprecipitated. 5. A large quantity of RNA should be diluted into several aliqouts to avoid freeze/ thawing unnecessarily. 6. To check the quality of the RNA, an OD 260,280ratio of b1.7 is acceptable. Values of ~1.7 may indicate protein contamination. 7. It is extremely important that the riboprobe template IS completely linearized prior to transcription. 8. Reagents used for template preparation must be RNase-free. Contaminated or dirty template stocks are a common cause for low incorporation. 9. Make sure that the nucleotlde used in the riboprobe transcription reaction is a ribonucleotide and not a deoxyribonucleotide. 10. CTP and UTP are recommended for radiolabeling riboprobes. The choice of which one depends on the number of sites of incorporation for each nucleotide: the more sites, the higher the specific activity of the probe. However, if the number of sites is too great, the radlolabeled RNA backbone may be unstable and probe degradation may occur. 11. In some procedures requiring the use of a riboprobe, a full transcription reaction is not necessary In such cases, a half-reaction (i.e., half the normal volume of reagents in a total reaction volume of 12.5 pL) may be used. The sample can then be DNase-treated and purified just as with a full reaction, but make sure the sample volume is 70 pL before loading it on the column. 12. Some protocols warn against the use of even the slightest excess of polymerase in an RNA transcription reaction, because of the possibdity of nonspecific transcription initiation at other promoter sites in the plasmld. However, this warning may be unfounded, since newer plasmids can contam multiple RNA polymerase promoters on the same strand of DNA without causing problems with transcription. 13. The main source of background hybridization in techniques that use riboprobes, such as the RNase protection assay, may be the incomplete digestion of the DNA

lredale et al.

14.

15.

16.

17.

18. 19. 20.

2 1. 22. 23.

template following the transcription reaction. Some of the newly synthesized probe may anneal to the complementary portion of the DNA template and, thus, render that region of the plasmid resistant to DNase treatment. This DNA would then be column-purified with the riboprobe and, during a hybridization reaction, would compete with mRNA for the probe. Resultant DNA-RNA hybrids would be resistant to RNase digestion and would increase the background of RPA samples on a polyacrylamide gel. If such a scenario is suspected, then the following modification should be made to the riboprobe transcription procedure: Following the labeling reaction, the sample should be heated for 10 min to 95’C for 5 min to denature DNA-riboprobe hybrids. Following quick cooling on ice for 10 s, DNase could then be added in the usual manner. Repeated freeze-thawing of reagents and synthesized riboprobes should be avoided, Aliquot materials in volumes that will not be freeze-thawed more than 3-5 times. In the random prime labeling technique, it is very important to determine the specificity of the DNA fragment to be labeled. The lack of probe specificity can cause unforeseen background hybridization problems. A search through GenBank would be useful. Another cause of high background hybridization when random prime probes are used is the presence of vector sequences in the fragment of interest. A DNA fragment with vector sequence at its ends should be digested at internal restriction sites to remove this unwanted DNA. The DNA fragment of interest must then be gel purified, making sure that the cleaved ends do not comigrate with it. The purification of multiple bands may result in hybridization to inappropriate target mRNAs. Low incorporation of radioactivity is generally caused by the quality of the punfied DNA. Although the purified DNA may appear clean on an agarose gel, impurities may be inhibiting the Klenow fragment. If this happens, a quick clean up of the DNA may solve the problem. Spin columns or phenol-chloroform extraction and ethanol precipitation are recommended. Wash flasks, gel casters, combs, and gel apparatus m 6% H,Oz for 20 min, followed by a quick rinse in DEPC-treated water to ensure that they are RNase free prior to use. If feasible, a gel apparatus and tank should be dedicated solely for RNA gels. RNA sample amounts: The amount of RNA necessary for message detection is dependent on the relative abundance of the target mRNA. Rare messages may require the isolation of mRNA in order to detect a signal. This is often the case with G protein-coupled receptors Hence, RPAs are commonly used, instead of Northern blot analysis, because of then enhanced sensitivity. The outermost lanes on either side of the gel should not be used if at all possible. RNA in these lanes tend not to transfer as well. Running the gel too fast may heat the agarose and distort the lanes of migrating RNA. Prior to removing the gel from the tank, it is advisable to cut off a comer of the gel for orientation purposes.

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24. It 1s important to remove all air bubbles between layers of materials being added to the capillary transfer apparatus. Air bubbles will prevent even transfer of RNA to nitrocellulose. A disposable IO-mL serological pipet works well to roll out the bubbles. 25. When prewetting mtrocellulose in 2X SSC, start with one comer and progress slowly until the entire piece is wet. This will prevent trapping of air in the membrane. 26. A hybridization oven is preferable to heat sealable bags for their ease m working with radIoactivity. There is less likely to be problems with leaking buffers and contamination of water baths or equipment. 27. If using hybridization tubes, be sure that the blots/mesh are not unraveling. Check this during the prehybridizatlon step. 28. The amount of labeled probe added to the hybridization buffer depends on the relative abundance of the target RNA. A good startmg point IS 1 x lo6 counts/min/mL buffer for abundant clones and 2-3 x IO6 countslminlml for rare messages. 29 Internal standards: Quantitation by Northern blot analysis may require the use of an internal standard to normalize results caused by uneven RNA loading or transfer. It is critical to first determine if the internal standard mRNA target is regulated by the paradigm being tested. Several riboprobe templates are available, including actin and cyclophillin (Ambion [Austin, TX]). If the size of the Internal standard transcript is different from the target RNA, it is more efficient to hybridize both probes at once. However, riboprobes and random prime labeled DNA probes cannot be used together, because they require different hybridization temperatures. Therefore, If the target probe and the internal standard probe are not of the same type, they must be run separately It is important to remember that often the level of RNA encoding the commonly used internal standards far exceeds the level of the target RNA, especially if studying G protein-coupled receptors. It is recommended that the internal standard probe be labeled to a lower specific activity than the tar&t probe. 30. It is important to preheat the wash buffers to temperature prior to addition to the blot. This will ensure that the appropriate wash conditions have been reached. 3 1, The wash protocol outlmed here is just a starting point. Additional or longer washes may be necessary. 32. Before purifying the riboprobe, remove 1 pL and determine counts/mm. Column purify probe and determine counts/min. Calculate percent mcorporatlon of radiolabeled nucleotide, and if it is less than approx 30%, discard probe. In general, efficient riboprobe labeling reactions should incorporate 50-70% of the label. A lower percent incorporation may indicate that the label has degraded, the template concentration is too low, and/or RNase contamination has been introduced into the labeling reaction. 33. It is critical that the probe be added to samples so that it is m vast excess of the mRNA to which it is to hybridize. Preliminary RPAs should be run on samples containing equal amounts of total RNA and increasmg amounts of probe, until a probe concentration is found that no longer yields an increased signal on an auto-

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35.

36. 37.

38. 39. 40.

41. 42.

43.

44. 45.

Iredale et al. radiogram. For most G protein-coupled receptors, 1 x 105 counts/mm of high specific activity probe should be sufficient. For end-labeling DNA size markers, the DNA must be digested with a restriction enzyme that leaves a 5’ overhang or blunt end; the correct radiolabeled nucleotide must be used. Klenow fills in a 5’ overhang with a radiolabeled nucleotide that IS complementary to the overhang. For example, a Hind111 digest leaves a 3’ TTAA 5’ overhang, and Klenow adds radiolabeled 5’ AA 3’ to the complementary strand. For enzymes that leave a blunt end, Klenow replaces the nucleotide at the 3’ terminus of the restriction site. For example, Hoe111 leaves a 5’ GG 3’ blunt end, and Klenow replaces the second G with a radiolabeled G. When pouring gels using the Bio-Rad Mini-PROTEIN II gel casting stand, apply a thm film of petroleum Jelly to each silicone gasket to ensure that no leaking occurs. Solutions for making polyacrylamide gels need not be DEPC-treated. Expect 8% gels to run for approx 70-90 min before the xylene cyan01 dye front reaches the bottom of the gel. To prevent the apparatus from heating up, which could cause smiling of the sample fronts, precool the 1X TBE to 4°C before adding to the chamber. 6% gels yield satisfactory results and are faster to run, but 8% gels yield nottceably sharper bands. It is important not to premix 10% SDS with Proteinase K before adding them to RNase-treated samples, because such concentrated SDS may denature the protease. DNA and RNA molecules of the same size electrophorese at different rates RNase-protected RNA fragments are approx 5-l 0% smaller m length than a DNA size marker traveling at the same rate. The half-life will vary, depending on the particular species of interest; therefore, these times are merely a guideline. Plot time (x-axis) agamst log % of control (v-axis) for both control and agonisttreated cells. The half-life (50% of control) can be derived by extrapolatmg from the graph (most computer programs will perform this function. Alternatively, the half-life can be determined from the slope of the line, using the formula: half-life = In 2/k, where In 2 = 0.693 and k = -slope of the line (see ref. IS). The lower the amount of DNA loaded per lane, the better the recovery. Unfortunately, it is necessary to cut large quantities of plasmrd m order to obtain sufficient amounts of cDNA (at least 30 pg for SIX samples) to bmd the nascent RNA transcripts from the nuclear run-on assay. Therefore, in order to keep the number of lanes to a practical number, it also becomes necessary to load larger amounts of cDNA per lane (20-40 pg of total DNA/lane). Handle the nylon with blunt forceps and gloved hands. It is a good idea to have an internal standard that can be used to normalize the results. This needs to be a gene that is ubiquitous and IS unaffected by the experimental conditions under observation, Cyclophilin and actin are commonly used examples; however, both can be affected by certain treatments. Linearized vector (minus the cDNA of interest) serves as a useful second control.

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46. Use a colored pencil to mark the location on the membrane of the slots containing cDNA and also the orientation, of the blot, since it is very difficult to see anything after the membrane has dried. 47. It is very important to use DEPC-treated water to make all the solutions, in order to minimize the possibility of RNase contamination. 48. It is important to keep the nuclei free from RNase contamination. DEPC-treat the Dounce homogenizers, pestles, and centrifuge tubes before use. 49. Use a 5-mL transfer pipet to layer the homogenate. Hold the centrifuge tube on a slight angle and drip the homogenate down the wall of the tube. 50. The pellet can be quite hard to resuspend; however, it is necessary for later stages in the nuclear run-on assay that the nuclear suspension is as homogeneous as possible. Therefore, take time to resuspend the pellet well by flicking the tube and pipeting the pellet up and down. The nuclei will withstand a certain amount of stress and taking the time at this point will alleviate unnecessary operator stress later in the assay. 51. Use 2-mL Eppendorf tubes with screw caps and 0 rings to reduce radioactive contamination. 52. The nuclei need to be passed through the needle at least three times to ensure complete shearing. Considerable care should be taken during this procedure to avoid aerosolizing radioactivity. 53. On average we try to add at least 12 x 1O6counts/mm/tube. The higher the amount of radiolabeled RNA added, the better the hybridizations, assuming that the overall level of RNA synthesis is not changing as a function of cell state The assumption might not always be valid and should therefore be examined prior to embarking on new experimental conditions. 54. Stop the second wash when the radioactivity is approx 200 counts/min (estimate using a Geiger counter). This is usually after 2-4 mm of the second wash; therefore, check the blots every 2 min to prevent over-washing.

Acknowledgments This work is supported by USPHS

grants MH4548

1, MH53 199, and 2 PO1

MH25642, and by a Veterans Administration National Center Grant for PTSD, VA Medical Center.

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13. Morgan, J. 1. and Curran, T. (1991) Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. Annu Rev. Neurosci. 14,42 l-45 1, 14. Armstrong, R. C. and Montminy, M. R. (1993) Transsynaptic control of gene expression. Annu Rev Neurosci. 16, 17-29. 15. Rodgers, J. R., Johnson, M. L., and Rosen, J. M. (1985) Measurement of mRNA concentration and mRNA half-life as a function of hormonal treatment. Methods Enzymoi. 109,572-592.