Prolactin Receptor

Prolactin Receptor Vincent Goffin* and Paul A. Kelly INSERM Unit 344, Faculty of Medicine Necker, 156 rue de Vaugirard, Paris, Cedex 15, 75730, France * corresponding author tel: ‡33-1-40615616, fax: ‡33-1-43060443, e-mail: [email protected] DOI: 10.1006/rwcy.2000.14012.

SUMMARY The prolactin receptor (PRLR) exists in all vertebrates in many isoforms, soluble or membranebound. It is expressed in a wide variety of tissues, and is responsible for the transmission of almost 300 distinct functions of its ligand, prolactin. This hormone activates the prolactin receptor by inducing its homodimerization, the first step required for triggering signaling cascades. No other membrane chain is required for signaling. Isoforms of prolactin receptor that vary in the length and sequence of the cytoplasmic tail have been identified; These variations account for the different signaling properties of the different isoforms. The JAK/STAT pathway (and mainly JAK2/STAT5) is the major signaling cascade, though many other proteins are also activated such as the MAP kinase pathway. Target genes are multiple, and confer to prolactin its numerous properties, such as lactogenic actions (milk protein gene induction) and mitogenic or antiapoptotic effects. Although no genetic abnormality has been linked to the PRLR gene, the involvement of this receptor in breast cancer has been proposed.

BACKGROUND

Discovery The prolactin receptor (PRLR) was identified in the early 1970s as a specific, high-affinity, saturable membrane-anchored protein, binding both prolactin and human growth hormone (Posner et al., 1974). It was first identified in liver, mammary gland, and reproductive organs from mammals, and later in many other tissues throughout all vertebrates, including fish. The original cloning of the cDNA encoding

the rat PRLR (short isoform) was reported in the late 1980s (Boutin et al., 1988).

Alternative names The prolactin receptor is also referred to as the `lactogen' receptor, or as the receptor for lactogenic or luteotropic hormones, two other names of prolactin. These names can be misleading, however, since the term `lactogenic' refers to the biological activity of milk production, whereas the prolactin receptor is responsible for the transduction of many nonlactogenic signals, e.g. mitogenic. In view of the wide spectrum of biological activities of prolactin, `prolactin receptor' is probably the most appropriate name for this protein.

Structure Like all class 1 cytokine receptors, the PRLR is a single-pass transmembrane chain, with the N-terminus outside the cell. In contrast to many cytokine receptors, for example the IL-2 receptor or IL-6 receptor families, the active form of the PRLR does not appear to involve any chain other than the PRLR itself. Depending on the species, there are many isoforms of the PRLR. These only vary in the length of the cytoplasmic domains, and hence in their signaling properties. In mammals, the overall length of the PRLR varies from  200 amino acids for the soluble binding protein up to  600 residues for the long membrane isoforms. Within the cytokine receptor superfamily, the growth hormone receptor (GHR) is undoubtedly the closest member to PRLR, with respect to its structure, its signaling properties, and its ligand.

1548 Vincent Goffin and Paul A. Kelly

Main activities and pathophysiological roles Prolactin was originally isolated based on its ability to stimulate mammary development and lactation in rabbits, and soon thereafter to stimulate the production of crop milk in pigeons (Stricker and Grueter, 1928; Riddle et al., 1933). Prolactin was also shown to be luteotropic, that is to promote the formation and action of the corpus luteum (Astwood, 1941). Subsequently, a number of additional activities have been associated with this hormone in various vertebrate species. In the now classical reviews by Nicoll and Bern (Nicoll and Bern, 1972; Nicoll, 1974), 85 different biological functions of prolactin were subdivided into five broad categories related to reproduction, osmoregulation, growth, integument, and synergism with steroids. Since the publication of these first reviews, numerous other biological functions of prolactin have been identified. Bole-Feysot et al. (1998) listed up to 300 functions or molecules activated by the PRLR, organized into the following categories: water and electrolyte balance, growth and development, endocrinology and metabolism, brain and behavior, reproduction, and finally immunoregulation and protection. Thus, despite the fact that prolactin remains historically linked to its actions in lactation and reproduction, the biological role of this hormone can no longer be restricted to these functions. Finally, although most circulating prolactin is of pituitary origin, emphasis has been given within the last few years to locally produced, nonpituitary prolactin. The wide distribution of the PRLR and the increasing number of tissues identified as prolactin sources (Ben-Jonathan et al., 1996) probably explains the unusually large number of functions of this hormone. Surprisingly, there is, to date, no disease known to be caused by a mutation of the PRLR, or even of its ligand. Hyperprolactinemia due to pituitary adenoma leads to reproductive disorders, especially in women, which can be treated either by drugs reducing prolactin secretion, or by surgical hypophysectomy.

GENE

Accession numbers The cDNAs encoding the PRLR have been cloned and sequenced in several vertebrates. GenBank: Human PRLR cDNA: M31661 (long isoform) Bovine PRLR cDNA: L02549 (long isoform)

Rat PRLR cDNA: M57668 (long isoform), M74152 (intermediate (or Nb2) isoform), M19304 (short isoform) Mouse PRLR cDNA: X73372 (long isoform), M22957/M22958/M22959 (short isoforms) Chicken PRLR cDNA: D13154 (long isoform) Fish PRLR cDNA: L34783 (long isoform) The original cloning of the short isoform PRLR was performed from rat liver (Boutin et al., 1988), then long isoforms were identified from mammary gland and ovarian cells (Boutin et al., 1989).

Sequence The coding sequence of the rat PRL receptor long isoform is given in Figure 1 (Shirota et al., 1990). The gene encoding the PRLR is unique, but several PRLR mRNAs resulting from alternative splicing are observed in (almost) all species studied thus far. Depending on the tissue and/or species considered, these various PRLR transcripts encode identical or different mature proteins. In humans, at least three mRNAs have been identified (2.8, 3.5, and 7.3 kb) that probably encode the sole long isoform; nevertheless, there is at least one study which reports the existence of a C-terminally truncated PRLR isoform in breast tumors (Clevenger et al., 1995). Conversely, in rodents, different mRNAs encode various isoforms differing in the length and composition of their cytoplasmic tail. These isoforms are referred to as short, intermediate, or long PRLR with respect to their size. In mouse, seven transcripts encode four different PRLR isoforms (one long, three short) (Buck et al., 1992). In addition to mRNAs encoding these membrane-anchored PRLR isoforms, alternatively spliced mRNA encoding soluble prolactinbinding protein (PRLbp) have been reported, such as in humans (Fuh and Wells, 1995).

Chromosome location and linkages The gene encoding the human PRLR is located on chromosome 5 (p13-14) and contains at least 10 exons for an overall length > 100 kb (Arden et al., 1990). In the mouse, the PRLR gene is > 120 kb and is located within a cluster of cytokine receptor loci on chromosome 15 (p12-13) (Gearing et al., 1993). The genomic organization of the mouse PRLR gene has recently been deciphered (Ormandy et al., 1998). It contains 13 exons, nine of which are shared by all PRLR isoforms, the last four encoding the C-terminal tail of the long isoform (exon 10) and the three short isoforms named PRLR S1 (exon 12), S2 (exon 11),

Prolactin Receptor 1549 Figure 1 Coding sequence of the rat long isoform of the PRLR. The sequence encoding the signal peptide is underlined and the sequence encoding the transmembrane domain is underlined and in italic.

and S3 (exon 13), the latter being homologous to the unique rat short isoform. Five different promoter regions have also been identified in the 50 UTR of the mouse PRLR gene (Ormandy et al., 1998). In the rat,

three promoters have been identified and tissuespecific usage has been demonstrated for two of them (Moldrup et al., 1996; Hu et al., 1998). Interestingly, at least in mouse, the genomic organization (coding

1550 Vincent Goffin and Paul A. Kelly sequences of exons) closely parallels the functional/ folding domains of the mature proteins; the two disulfides are encoded by exons 4 and 5, the WS motif by exon 7, the transmembrane domain by exon 8 and the Box-1 by exon 9 (Ormandy et al., 1998).

PROTEIN

Accession numbers SwissProt accession numbers for the PRLR precursors are listed in Table 1.

Sequence The sequence of the rat long isoform of the PRLR is given in Figure 2.

Table 1

Description of protein The PRLR protein is a glycosylated, single-pass transmembrane protein with the N-terminus in the extracellular space. It is synthesized as a precursor including a signal peptide of  19 to  24 amino acids. Within each species, different PRLR isoforms can be observed that have strictly identical extracellular (ligand-binding) domains and differ only by the length of their cytoplasmic tail (Figure 3). For example, the rat PRLR isoforms contain 291 (short), 393 (intermediate), or 591 (long) amino acids, and are identical until residue 260 (after Box-1). In SDSPAGE (western blots), the long PRLR migrates with an apparent size of 90±95 kDa due to posttranslational modifications, whereas the rat short PRLR appears as a doublet at 42±44 kDa. Of the other cytokine receptors, the growth hormone receptor (GHR) is closest to the PRLR in terms of protein structure, signaling properties, and ligands.

Accession numbers (SwissProt) of PRLR precursors (including signal peptides)

Species

Accession number

Isoform

Precursor

Mature

Human

P16471

Long

622 aa

598 aa

Bovine

Q28172

Long

581 aa

557 aa

Rat

P05710

Long

610 aa

591 aa

Mouse

Q08501

Long

608 aa

589 aa

Fish (tilapia)

Q91513

Long

630 aa

606 aa

Figure 2 The sequence of the rat long isoform of the PRLR. The signal peptide is underlined, the transmembrane domain is underlined and in italic.

Prolactin Receptor 1551 Figure 3 Schematic representation of soluble (human) and membrane (rat) isoforms of the PRLR. Although the mechanism of PRLbp generation remains unclear (alternative splicing or proteolysis or both), an mRNA encoding a soluble PRLbp of 206 amino acids has been isolated in the human breast cancer cell line BT474. In a given species, all forms have an identical extracellular ligand-binding domain. Subdomain D1 contains two pairs of disulfide-bonded cysteines (C±C) and subdomain D2 the WS motif (green box), two characteristic features of the cytokine receptor superfamily. Box-1 (orange box) is found in the cytoplasmic domain of all membrane isoforms. In rat, the intermediate PRLR (only found in Nb2 cells) differs from the long isoform by a 198 amino acid deletion in the cytoplasmic domain (amino acids 323±520). Otherwise, the short PRLR is identical to both other isoforms up to residue 260, after which its sequence differs (light blue box). Cytoplasmic tyrosine residues are indicated. (Full colour figure may be viewed online.)

The extracellular domain of the PRLR is a typical cytokine extracellular domain, composed of a sequence of  210 amino acids referred to as the cytokine receptor homology (CRH) region. In contrast to many other cytokine receptors, there is no additional domain. The PRLR extracellular domain is divided into two subdomains of  100 amino acids (referred to as D1 and D2), both showing analogies with the fibronectin type III module (Kelly et al., 1991). Two highly conserved features are found in all extracellular domains of PRLR isoforms: the first is two pairs of disulfide-linked cysteines in the N-terminal subdomain D1 (Cys12±Cys22 and Cys51±Cys62 in human PRLR), and the second, a characteristic

feature of cytokine receptors, is the `WS motif' found in the membrane proximal region of subdomain D2. These features are required to obtain fully active receptors: mutation of cysteines leads to misfolded proteins with impaired ligand-binding properties, whereas mutation of the WS domain alters cell trafficking of the receptor (impaired export to cell surface). In addition to these features, two tryptophans (Trp72 and Trp139 in human PRLR) conserved in the PRLR and in the closely related growth hormone receptor are presumably important for binding prolactin. The transmembrane domain is 24 amino acids long (residues 211±234 in human PRLR). The possible involvement of this region (or of any crucial amino acid within this domain) in the functional activity of the receptor is unknown. The cytoplasmic domain is the only region which distinguishes PRLR isoforms. The cytoplasmic domain can be very short (57 amino acids in the short rat PRLR), and attains 357 amino acids in the long PRLR. The intracellular domain is devoid of any intrinsic enzymatic (tyrosine kinase) activity. Two regions, called Box-1 and Box-2, are conserved features (Bole-Feysot et al., 1998). Box-1 is a membraneproximal region composed of eight amino acids highly enriched in prolines and hydrophobic residues (amino acids 243±250 in rat PRLR). Due to the particular structural properties of proline residues, the conserved P±X±P (X ˆ any amino acid) motif within Box-1 is assumed to adopt a consensus folding specifically recognized by transducing molecules. The second consensus region, Box-2, is much less conserved than Box-1 and consists of a succession of hydrophobic, negatively charged then positively charged residues (amino acids 288±298 in rat). While Box-1 is conserved in all membrane PRLR isoforms, Box-2 is not found in short isoforms. Finally, two motifs involved in receptor internalization, namely a dileucine and a predicted turn, have been identified in the rat short PRLR. It is noteworthy that apart from Box-1, no consensus folding domain (SH2, SH3, PTB, WW, etc.) has been identified within the cytoplasmic domain of the PRLR. The three-dimensional structure of a genetically engineered human PRLR extracellular domain has been determined by crystallographic analysis (Somers et al., 1994) (Figure 4). Each D1 and D2 subdomain folds in seven strands forming a sandwich of two antiparallel sheets. Both subdomains are linked by a small four-residue polypeptide. This structure is the conformational paradigm of the CRH domain (Bazan, 1990) and is shared by the growth hormone receptor, the EPO receptor, and the IFN receptor.

1552 Vincent Goffin and Paul A. Kelly Figure 4 Ribbon representation of the threedimensional X-ray structure of a monomer of the human PRLR extracellular domain. The extracellular domain folds in a sandwich formed by two antiparallel sheets (see text). N- and C-terminal ends are indicated by N and C, respectively. This figure was kindly provided by Dr A. M. de Vos.

residues, including the last tyrosine which was shown to be functionally important in other mammalian PRLRs (see Signal transduction section). In addition to these species-specific variations, it has been observed that the N-linked glycosylation sites found in the PRLR extracellular domain are not strictly conserved in all species (Buteau et al., 1998).

Affinity for ligand(s)

To the best of our knowledge, no structural data have been reported yet for the cytoplasmic domain of any cytokine receptor, including the PRLR.

Relevant homologies and species differences The variability of PRL receptors has more to do with the existence of various isoforms within a given species than with any interspecies differences. However, two atypical cases can be cited. First, avian PRLRs are particular in that their extracellular domain is duplicated and contains two highly homologous CRH domains; the additional N-terminal module seems, however, to have no functional role (Gao et al., 1996). Second, in cervine and bovine PRLR, the C-terminal tail is truncated by  35

The PRL receptor binds to at least three types of ligands: prolactin, primate growth hormone, and placental lactogen (PL) which is synthesized by mammal placenta and is thus not found in lower vertebrates. These ligands belong to a hormone family termed the PRL/GH/PL family (Goffin et al., 1996b). Although growth hormone binds to its specific receptor, growth hormone from primates (human, monkeys) is able to bind to the PRLR as well. There is currently no specific receptor identified for PLs, which binds to the PRLR and/or the growth hormone receptor, depending on the species considered (Gertler et al., 1997). At least two parameters can modulate the reported affinities of the PRLR for its ligands. First, it is usually observed that the soluble binding protein (PRLbp) has a higher affinity ( 10 times) than the membrane-bound PRLR for a given ligand (PostelVinay et al., 1991), and the length of cytoplasmic tail also influences the overall affinity, although to a lesser extent (Ali et al., 1991). Second, the affinity of the PRLR will vary depending on the type and species of origin of the ligand considered. For example, the affinity of human growth hormone, but not of PRL, for the PRLbp is modulated by 8000-fold depending on the zinc concentration (Cunningham et al., 1990), an effect which is explained by the fact that two amino acids within human growth hormone coordinate one zinc ion together with two residues of the human PRLbp (Somers et al., 1994). In conclusion, depending on these parameters and cross-species variation, the affinity of the PRLR for its ligands is usually in the range of Kd=10ÿ9 to 10ÿ10 M. The binding of these ligands to the PRLR is the first step of receptor activation. Several studies have shown indirectly that the PRLR is activated by dimerization (Goffin et al., 1996b) (Figure 5), which involves two regions (so-called binding sites 1 and 2) of the ligands, each interacting with one molecule of PRLR. Even though crystallographic analysis of the PRL/PRLbp complex is lacking, it is anticipated from the three-dimensional structure of the closely-related hGH/hGHBP complex (De Vos et al., 1992) that both binding sites interact with virtually overlapping epitopes within the receptor. Thus far, no accessory

Prolactin Receptor 1553 Figure 5 PRLR activation by PRL-induced dimerization. Hormone binding to PRLR is sequential. First, the hormone interacts with the receptor through its binding site 1, forming an inactive H1 : R1 complex. Then, the hormone binds to a second receptor through its site 2, which leads to receptor homodimerization and formation of an active H1 : 2R2 complex. Hormone analogs whose binding site 2 is sterically blocked are unable to induce receptor homodimerization and are thus inactive; since they still bind to the receptor through site 1, they behave as antagonists of wild-type hormones.

membrane protein has been shown to be required for PRLR signaling.

Cell types and tissues expressing the receptor PRL receptors have been identified in a wide range of cells and tissues. In addition to the previously known PRL targets, such as mammary gland or reproductive organs, many other organs have been found to express the PRLR. An exhaustive list is provided in Table 2 (Bole-Feysot et al., 1998). With respect to in vitro cell cultures, the Nb2 rat lymphoma is one of the preferred cell systems used to investigate PRLR-related events. First, this cell line expresses 12,000 PRL receptors per cell, which allows easy study of PRLR signaling (see below). Second, the proliferation of Nb2 cells is induced by very small amounts of lactogens (<1 ng/mL) which makes this cell line the most widely used bioassay for quantifying the lactogen content of physiologic fluids or of cellconditioned media, or for estimating the biological properties of mutated proteins of the PRL/GFH/PL family. In addition to the Nb2 cell line, mouse mammary epithelial cells such as HC-11 or human mammary tumor cell lines such as T-47D or MCF-7 also express detectable amounts of PRLR.

Regulation of receptor expression The level of expression of the PRLR varies from  10 to  2000 fmol/mg of membrane protein. The expression of short and long forms of receptor have been

shown to vary as a function of the stage of the estrous cycle, pregnancy, and lactation. However, the hormonal regulation is different depending on the target organ, and due to the extremely wide distribution of the PRLR, it is currently difficult and premature to propose a general overview of the regulation of PRLR expression. Since no recent review on this theme is available, it is probably best to do a MedLine search for the organ of interest. In the mammary gland, where the long form is predominant, PRLR levels increase upon lactation, and in breast tumor cells its regulation is crossregulated with that of sex steroid hormone receptors (Ormandy et al., 1997b). In rat liver, the short PRLR isoform is predominant and is increased by estrogens. In the prostate, PRLR levels are increased by testosterone, and decreased by estrogens. In the mouse ovary, PRLR expression also varies during pregnancy (Clarke et al., 1993), and in the human endometrium, the PRLR expression is upregulated during the secretory phase of the menstrual cycle (Jabbour et al., 1998). Prolactin is able to both up- and downregulate its receptor, the latter process probably being due to an acceleration of internalization of hormone/ receptor complexes. The effect of prolactin on its receptor is also a function of the hormone concentration and time of exposure of the tissue. Growth hormone is also able to upregulate the PRLR (Kelly et al., 1991).

Release of soluble receptors Soluble receptors are naturally observed, but whether they result from alternative splicing or membrane

1554 Vincent Goffin and Paul A. Kelly Table 2 Distribution of prolactin-binding sites in vertebrates

Table 2 (Continued )

Central nervous system

Kidney

Brain

Crop sac (birds)

Cortex

Bladder (fishes, reptiles, amphibians)

Hippocampus

Lymphoid tissue

Choroid plexus Striatum

Nurse cells

Cochlear duct

Epithelial cells Lymphocytes

Hypothalamus

T

Astrocytes

B

Glial cells

Macrophages

Retina

Ganglia

Olfactory system

Intestinal cells

Ganglia

Reproductive system (female)

Ova

Intermediate lobe

Granulosa cells Thecal cells Corpus luteum (luteal cells)

Epidermis Hair follicle

Oviduct

Sweat glands Bone tissue

Mammary gland

Chondrocytes

Epithelial cells

Cartilage

Milk

Osteoblasts

Tumors

Gills (fishes and larval amphibians)

Uterus (endometrium)

Lung Heart

Ovary

Anterior lobe

Adrenal cortex Skin

Spleen Thymus

Corpus callosum

Pituitary

Cortex

Cardiac muscle

Placenta

Atria Skeletal muscle

Amnion Reproductive system (male)

Testis

Adipocytes (birds)

Germ cells

Brown adipose tissue

Spermatozoa

Liver

Hepatocytes

Leydig cells

Kupffer cells

Sertoli cells

Submandicular gland

Epididymis

Submaxillary gland

Seminal vesicle

Pancreas

Islet of Langerhans

Gastrointestinal tract

Esophagus Stomach Intestine Duodenum Jejunum Ileum Colon

Prostate

receptor proteolysis or both is still to be clearly established (Postel-Vinay et al., 1991). To our knowledge, no protease has been identified, and few studies report the existence of alternatively spliced mRNAs encoding a PRLbp (Fuh and Wells, 1995). As for the GHBP, some species-specificity might be observed in the mechanism of binding protein release. Soluble

Prolactin Receptor 1555 isoforms are identical to the extracellular, ligandbinding domain of the PRLR, with minor differences regarding truncation of the last few C-terminal amino acids. The physiological role of binding proteins remains unclear. It is usually assumed that the formation of PRL/PRLbp complexes enhances the half-life of the hormone in blood circulation. Moreover, since activation of the PRLR occurs by ligand-induced dimerization, such complexes may interfere with signaling properties of membranebound PRLR if receptor heterodimers (one membrane-bound, one soluble) were to be formed (Lesueur et al., 1993).

SIGNAL TRANSDUCTION All of the actions mediated by the PRLR result from its interaction with any of the natural ligands, which leads to receptor dimerization and activation of cascades in the intracellular space. Nb2 cells are a rat lymphoma (pre-T cell line) which expresses 12,000 PRLRs per cell and proliferates in the presence of very low amounts of lactogens (Gout et al., 1980). Although the PRLR expressed in Nb2 cells is a mutated form of the long PRLR (so-called intermediate isoform), lacking 198 amino acids within the cytoplasmic domain (Ali et al., 1991) (Figure 3), this cell line remains one of the preferred models to study prolactin actions with respect to signal-transducing molecules that are activated. It is noteworthy that there is currently no clear picture whether the mutation of the Nb2 PRLR has any incidence on signaling cascades that are triggered by this truncated PRLR compared with the long isoform. In the following section, only a brief overview of PRLR signaling is proposed (for further discussions and references, see Hennighausen et al., 1997; Bole-Feysot et al., 1998; Clevenger et al., 1998; Yu-Lee et al., 1998).

binding, contrary to what has been observed for the growth hormone receptor. The PRLR±JAK2 interaction requires the Box-1 domain, which does not preclude the involvement of additional residues towards the C-terminus. Since Box-1 is conserved in all PRLR isoforms, signaling differences between the different isoforms are anticipated not to be directed by the ability to associate/activate the kinase. The most C-terminal proline of Box-1 (Pro250 in rat PRLR) is critical for association with and subsequent activation of JAK2. Whether the interaction between JAK2 and the PRLR is direct or involves an intermediate protein (adapter) is currently unknown. To the best of our knowledge, no mutational study aimed at mapping the region of JAK2 interacting with the PRLR has been reported yet, although investigation of the structural features required for the formation of other cytokine receptors/JAK complexes suggest the involvement of the N-terminal region of the kinase in the interaction with cytokine receptors. Other kinases have also been reported to be associated with/activated by the PRLR in the rat T lymphoma Nb2 cell line, such as the serine/threonine kinase Raf-1 (Clevenger et al., 1994) and the Src tyrosine kinase family member, Fyn (Clevenger and Medaglia, 1994). Association of the PRLR with Src, the prototype member of the Src kinase family, has also been reported after prolactin stimulation in lactating rat hepatocytes (Berlanga et al., 1995). Prolactin induces a rapid tyrosine phosphorylation of the 85 kDa subunit of the phosphatidylinositol (PI) 3'-kinase which, with IRS-1, appears to associate with the PRLR in a prolactin-dependent manner. For these numerous kinases, however, the sites of association on the receptor, their upstream and/or downstream effectors, as well as the biological functions/ pathways in which they are involved are poorly understood.

Associated or intrinsic kinases

Cytoplasmic signaling cascades

The PRLR is devoid of any intrinsic enzymatic activity. In 1994, JAK2 was identified as the Janus kinase associated with the PRLR (Rui et al., 1994). Although the involvement of JAK1 has also been proposed in the particular context of mouse lymphoid BAF/3 cells transfected with the PRLR (DusanterFourt et al., 1994), and in addition, the possible interaction between JAK2 and JAK3 in avian PRLR signaling has been suggested (Gao et al., 1996), JAK2 is unambiguously the major PRLR-associated Janus kinase. JAK2 is constitutively associated with the PRLR, i.e. its recruitment is not induced by ligand

As the paradigm of cytokine receptor signaling, the JAK/STAT signaling pathway is the most widely described cascade for the PRLR (Figure 6). Activation of JAK2 by the PRLR occurs very rapidly after hormonal stimulation (within 1 minute), suggesting that this Janus kinase occupies a central and very upstream role in the activation of several signaling pathways of the PRLR. As expected, PRLR mutants unable to associate with JAK2, such as Box-1-deleted mutants, are unable to trigger tyrosine phosphorylation cascades and downstream activation of PRLR-responsive reporter genes.

1556 Vincent Goffin and Paul A. Kelly Figure 6 Schematic representation of the PRLR receptor signaling pathways. Long and short isoforms of rat PRLR are represented. PRLR activates STAT1, STAT3 and, mainly, STAT5. Interaction of STAT5 with the glucocorticoid receptor (GR) has been reported. Whether the short PRLR isoform activates the STAT pathway is currently unknown. PRAP seems to interact preferentially with the short PRLR. The MAP kinase pathway involves the Shc, Grb2, Sos, Ras, Raf cascade and is presumably activated by both PRLR isoforms. Connections between the JAK/STAT and MAP kinase pathways have been suggested. Interactions between receptors and Src kinases (e.g. Fyn), SHP2, IRS-1, PI-3 kinase and other transducing molecules remain unclear.

Heterodimerization of the short and the intermediate PRLR cytoplasmic tails produces complexes unable to stimulate JAK2 autophosphorylation (Chang et al., 1998), whereas both can associate with and activate the kinase in the context of their respective wild-type receptor. This observation might be of importance in the physiological context since in species known to express different PRLR isoforms (e.g. rat), all tissues express both isoforms, although in varying ratios. In transfected cells, the short PRLR functions as a dominant negative isoform, inhibiting the activation of milk protein gene transcription by the receptor complex through heterodimerization (PerrotApplanat et al., 1997). All rat PRLR isoforms are able to activate JAK2, which in turn phosphorylates the receptor on tyrosines, with the exception of the short isoform which does not undergo tyrosine phosphorylation in spite of the presence of four tyrosines in its cytoplasmic domain. In rat intermediate and long PRLR isoforms, the most C-terminal tyrosine is one major target of JAK2. This is not an absolute rule, however, since activation of the IRF-1 promoter (see below) requires

other tyrosines as well (Wang et al., 1997; Yu-Lee et al., 1998), and several studies suggest that tyrosines other than the C-terminal can be phosphorylated in the long PRLR as well (Pezet et al., 1997; Bole-Feysot et al., 1998; Mayr et al., 1998). Accordingly, the natural C-terminal truncation of the cytoplasmic tail (including the last tyrosine) in bovine and cervine PRLR is not detrimental to receptor activity, indicating that alternative intracellular tyrosines can be used (Jabbour et al., 1996). To the best of our knowledge, no correlation between tyrosines that are preferentially phosphorylated by JAK kinases and their surrounding amino acids has been identified. Despite the possible redundancy of tyrosine phosphorylation within the long PRLR isoforms, the most C-terminal tyrosine appears critical for stimulating reporter genes containing milk protein gene promoters and for cell proliferation. Mutation of the C-terminal tyrosine within the intracellular domain of a single intermediate PRLR (Nb2 form) involved in a dimerized complex strongly decreases prolactininduced proliferation of BA/F3 cells (Chang et al., 1998). Thus, not only is this residue important, but it

Prolactin Receptor 1557 also needs to be present on both chains of the PRLR dimer. The PRLR phosphorylated tyrosines serve as docking sites for STAT proteins (signal transducer and activator of transcription). Accordingly, it is noteworthy that the (in)ability of the short PRLR isoform (which is not phosphorylated on tyrosine) to activate STAT proteins remains to be definitely established. Three members of the STAT family have been thus far identified as transducer molecules of the PRLR (long and intermediate): STAT1, STAT3, and, mainly, STAT5. STAT5, initially referred to as mammary gland factor (MGF) was identified from sheep mammary gland, the major target tissue of prolactin (induction of milk protein gene expression). From mutational studies, the phosphorylated Cterminal tyrosine is a good candidate for recruiting STAT5 (Figure 6), even though involvement of other tyrosines cannot be disregarded (Bole-Feysot et al., 1998; Mayr et al., 1998). Two homologous genes encoding STAT5 (namely A and B) have been identified, and both can be activated by prolactin, although with different kinetics (Kirken et al., 1997). In addition to being tyrosine phosphorylated by JAK2, STAT5A/B are also phosphorylated on serine, but the kinase involved remains to be identified (Kirken et al., 1997). A recent report suggests that two pathways are involved in serine phosphorylation of STAT5, one that is prolactin-activated and MAP kinase inhibitorsensitive, and one that is constitutively activated and MAP kinase inhibitor-insensitive (Yamashita et al., 1998). Finally, Yu-Lee and coworkers have shown recently that STAT5A and STAT5B exert an inhibitory effect on PRL-inducible IRF-1 promoter activity, and these authors have proposed this inhibition to involve squelching by STAT5 of a factor that STAT1 requires to stimulate the IRF-1 promoter (Luo and Yu-Lee, 1997; Yu-Lee, 1997). STAT1 and STAT3 are also activated by the PRLR. The region(s) of the PRLR required for activation of these STATs are less well documented. In the Nb2 PRLR, the C-terminal and middle tyrosines (Tyr382 and Tyr309) are proposed to bind STAT 1 (Wang et al., 1997). In the context of the growth hormone receptor, it has been hypothesized that phosphotyrosine(s) of JAK2 could also bind to STAT3, in agreement with the presence of the consensus STAT3-binding sites in the kinase. Although such an interaction does not preclude the possible occurrence of interactions with the receptor, this hypothesis remains to be tested in the context of STAT activation by the PRLR. The recent discovery of a family of proteins downregulating the activation of the JAK/STAT pathway has greatly helped our understanding of how

these activated (phosphorylated) proteins come back to their steady state after prolactin (and cytokine in general) stimulation. These proteins are named SOCS (suppressor of cytokine signaling) or CIS (cytokineinducible SH2), and JAB (JAK2-binding proteins) and contain at least five members (CIS-1, -2, -3, -4, and JAB). A recent study demonstrates that PRLRactivation of a STAT5-responsive reporter gene in transfected 293 cells is abolished by transfected CIS-3 and JAB (Helman et al., 1998). Interestingly, SOCS genes are target genes of the JAK/STAT pathway, which provides a direct negative regulation of this cascade (see below). The JAK/STAT is undoubtedly the major cascade triggered by the PRLR, as emphasized by the poor understanding of PRLR signaling before the Janus kinase family was discovered in the early 1990s. However, many other signaling proteins were found to be activated by the PRLR. The well-known MAP kinase pathway involves the Shc/SOS/Grb2/Ras/Raf/ MAP kinase cascade and this pathway has been demonstrated to be activated by the PRLR, including the short isoform, in various cell systems (Piccoletti et al., 1994; Das and Vonderhaar, 1996; Erwin et al., 1996). Whether activation of the MAP cascade requires JAK2, Fyn, Src or any other pathway is currently unknown. Although the JAK/STAT and the MAP kinase cascades were initially regarded as independent pathways, recent data rather suggest that these pathways are interconnected (Chida et al., 1998). Finally, other proteins that are activated by the PRLR can be listed, despite the fact that the cascade they are involved in remain poorly deciphered: the phosphatase SHP-2, the adapters IRS-1, IRS-2, and IRS-3, the PRLR-associated protein PRAP recently identified as a 17 -hydroxysteroid dehydrogenase/ 17-ketosteroid reductase (Nokelainen et al., 1998), the adapter Cbl, vav, and proteins linked to apoptotic pathway including Bax, Bcl-2 and Bag-1 (see referencecs in Bole-Feysot et al., 1998). Currently, however, these molecules are not considered as major signaling events triggered by the PRLR. Crosstalk with the tyrosine kinase (noncytokine) receptor for EGF has also been reported (Lange et al., 1998; Quijano and Sheffield, 1998).

DOWNSTREAM GENE ACTIVATION

Transcription factors activated When activated, STAT factors translocate to the nucleus, where they transactivate target gene

1558 Vincent Goffin and Paul A. Kelly promoters by binding to consensus DNA sequences. The interaction of STATs with other transcription factors has been reported. For example, the glucocorticoid receptor interacts with STAT5 and positively modulates gene transactivation (Stocklin et al., 1996, 1997). Interaction between STATs activated by the PRLR and other nuclear receptors, such as the progesterone receptor, has been demonstrated (Richer et al., 1998) or, as for the estrogen receptor, is anticipated from functional interference observed in vitro (e.g. transfected cells) but awaits experimental confirmation. Interaction between STAT1 and CBP300 (CREB-binding protein), which itself does not bind DNA but interferes with transcription factors through protein±protein interactions, has also been shown to enhance IRF-1 promoter activity. Co-operative interaction between NF1 and STAT5 on WAP gene expression has also been described (Li and Rosen, 1995; see Chida et al., 1998 and references therein).

Genes induced Due to the extremely wide spectrum of prolactin activities, an exhaustive listing of genes activated is beyond the scope of this chapter (Horseman and YuLee, 1994; Bole-Feysot et al., 1998). In the mammary gland, milk protein genes are obvious targets (caseins, lactoglobulin, whey acidic protein, etc.), and full activation of these genes requires other hormones (insulin, glucocorticoid) and extracellular matrix components. In liver, prolactin stimulates the transcription of early growth-related genes such as c-fos, c-jun, c-myc, c-src, IGF-1, and mid- and late G1 genes such as ornithine decarboxylase (ODC), heat shock proteins (hsp), or gfi-1. In Nb2 cells, prolactin activates expression of most of these genes, plus those of the transcription factor IRF-1, Bax, Bcl-2, and many cyclins. In a more general fashion, members of the CIS protein family, the downregulators of the JAK/STAT pathways, are target genes of PRLR signaling; however, the cell-specificity of this induction remains to be investigated (Helman et al., 1998).

Promoter regions involved Consensus DNA motifs specifically recognized by STAT complexes have been identified in the promoters of target genes. The motif termed GAS (for gamma interferon-activated sequence) was defined

using STAT homodimers and consists of a palindromic sequence TTTCxxxGAAA. This motif is found in the promoters of all STAT target genes. The specificity of the interaction between a particular STAT and a GAS motif found in a given target promoter has been proposed to depend, at least in part, on the center core nucleotide(s) (Ihle, 1996). Whether the synergism of STATs with other transcription factors involves DNA binding of the latter is not a general rule, and remains controversial for the glucocorticoid receptor (Lechner et al., 1997; Stocklin et al., 1997). The activation of identical STAT proteins by different cytokine receptors questions the mechanisms by which specificity of signaling pathways is achieved in response to a particular hormonal stimulation. Although several cytokines (e.g. EPO, GM-CSF, GH, PRL, IL-2, IL-3, and IL-5) activate the DNA-binding ability of STAT5 and/or transactivate the -casein luciferase reporter gene in vitro, -casein is only found in mammary epithelial cells and in cytotoxic T cells. Moreover, in mammary gland, activation of this gene promoter requires other lactogenic hormones such as insulin and glucocorticoid. This suggests that different STAT combinations and/or involvement of other signal transducers/transcription factors direct the specificity of the final response. This question has been recently investigated by Miyajima and coworkers who proposed that the activation of STAT5 involves complex interactions with other signaling pathways and requires integration of opposing signals from Ras (Chida et al., 1998).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors As emphasized by the phenotypes of prolactin knockout and PRLR knockout mice, milk production and reproductive properties are the functions that can obviously not be taken over by other hormones or cytokines, despite the likelihood that cytokines display some functional redundancy. Besides these actions, the extremely wide spectrum of prolactin activities must probably be regarded as a panel of functions that are modulated by, rather than unique to prolactin or its receptor.

Prolactin Receptor 1559

Phenotypes of receptor knockouts and receptor overexpression mice Knockout mouse models for the PRL gene (Horseman et al., 1997) and for the PRLR gene (Ormandy et al., 1997a) have been developed. Both exhibit the same general pattern of reproductive deficiencies. Several additional phenotypes have been reported for the receptor knockout. This may be related to the fact that knockout of the PRLR abolishes all activities naturally depending on this receptor, while knockout of the ligand prolactin does not prevent other potential ligands from binding and activating the receptor. Neither of these knockouts is lethal, indicating that the PRLR is not an absolute requirement for fetal development. The main phenotypes of PRLR knockout mice are linked to the sterility of double negative females, due to a failure of embryo implantation. Along with their impaired ability to lactate, these two phenotypes correlate well with the historically known functions of prolactin, referred to as the `lactogenic and luteotropic hormone'. Interestingly, heterozygous females are not sterile, but also show impaired lactation, which can be restored to normal level by successive pregnancies. Other phenotypes have recently been evaluated. Maternal behavior of double negative females towards foster pups is altered (Bridges et al., 1985; Lucas et al., 1998). Bone formation is also impaired (Clement-Lacroix et al., 1999), which was unexpected since the potential role, even indirect, of prolactin on bone cells had been ignored earlier. Despite studies on hypophysectomized animals indicating an immunomodulatory role of prolactin (Nagy and Berczi, 1991), no immunological phenotype was observed in either PRLR or PRL knockout mice. This suggests that PRL- or PRLR-deficient mice can probably compensate for the lack of receptor functions via redundancy of (an)other cytokine(s).

Human abnormalities To date, no disease has been linked to any genetic abnormality of the PRLR. This would suggest either that mutations have no detectable effect in vivo, or that such mutations might be lethal and, thereby, never detected. PRLR knockout mice are viable, so it is clear that the PRLR is not essential for survival, at least in this species. On the other hand, since there are important reproductive effects in females, this could explain the lack of genetic transmission. Hyperprolactinemia caused by hypersecretion of prolactin by pituitary adenomas usually leads to

various endocrine effects, with consequences on reproductive functions, especially in women. These patients are treated with dopamine agonists, the natural inhibitor of prolactin secretion, or alternatively they often undergo surgical removal of the adenoma. Prolactin is believed to be one of the factors favoring the proliferation of some tumors, such as breast cancer or prostate cancer. Mammary tumors express more PRLR than normal tissue (Touraine et al., 1998), and the local secretion of mammary prolactin has been suggested to stimulate cell proliferation in an autocrine/paracrine manner (Ginsburg and Vonderhaar, 1995). If this is true, prolactin could be carcinogenic in the mammary gland, which is clearly established in animal models of prolactin hypersecretion (Wennbo et al., 1997) but remains to be demonstrated in humans. Finally, prolactin has been shown to be increased and to affect a number of autoimmune states (BoleFeysot et al., 1998), such as systemic lupus erythematosus, acute experimental allergic encephalomyelitis, rheumatoid arthritis, adjuvant arthritis, and graftversus-host disease, for example as a marker of rejection in heart transplantation. Prolactin has also been suggested to be involved in the etiology of cystic fibrosis, although the precise mechanism remains unclear.

THERAPEUTIC UTILITY

Effect of treatment with soluble receptor domain To the best of our knowledge, no disease has been tentatively treated with soluble prolactin-binding protein. The binding protein could, however, bind human growth hormone, and thus have an effect on the half-life and potentially the biological activity of growth hormone as well.

Effects of inhibitors (antibodies) to receptors In vivo, we are not aware of any clinical trials involving anti-PRLR or anti-PRL antibodies. Disorders linked to prolactin synthesis are treated by lowering its secretion by dopamine. In vitro studies have shown that anti-human PRL antibodies can prevent proliferation of breast cancer cell lines induced by locally produced prolactin (Ginsburg and Vonderhaar, 1995), which might identify nonpituitary prolactin as a new target in breast cancer

1560 Vincent Goffin and Paul A. Kelly therapy. Since regulation of extrapituitary PRL is probably not controlled by dopamine, alternative strategies to dopamine agonists must be investigated to prevent the putative autocrine/paracrine effect of prolactin in mammary tumors. Prolactin antagonists have been generated by engineering prolactin mutants with highly impaired ability to induce PRLR dimerization (i.e. activation) (Goffin et al., 1996a, 1996b; Bole-Feysot et al., 1998). Such prolactin variants might be of use in future to counteract the proliferative effect of prolactin in breast tumors.

References Ali, S., Pellegrini, I., Edery, M., Lesueur, L., Paly, J., Djiane, J., and Kelly, P. A. (1991). A prolactin-dependent immune cell line (Nb2) expresses a mutant form of prolactin receptor. J. Biol. Chem. 266, 20110±20117. Arden, K. C., Boutin, J. M., Djiane, J., Kelly, P. A., and Cavenee, W. K. (1990). The receptors for prolactin and growth hormone are localized in the same region of human chromosome 5. Cytogenet. Cell Genet. 53, 161±165. Astwood, E. B. (1941). The regulation of corpus luteum function by hypophysial luteotrophin. Endocrinology 29, 309±319. Bazan, J. F. (1990). Structural design of molecular evolution of a cytokine receptor superfamily. Proc. Natl Acad. Sci. USA 87, 6934±6938. Ben-Jonathan, N., Mershon, J. L., Allen, D. L., Steinmetz, R. W. (1996). Extrapituitary prolactin: distribution, regulation, functions, and clinical aspects. Endocr. Rev. 17, 639±669. Berlanga, J. J., Fresno, V., Martin-Perez, J., and Garcia-Ruiz, J. P. (1995). Prolactin receptor is associated with c-src kinase in rat liver. Mol. Endocrinol. 9, 1461±1467. Bole-Feysot, C., Goffin, V., Edery, M., Binart, N., and Kelly, P. A. (1998). Prolactin and its receptor: actions, signal transduction pathways and phenotypes observed in prolactin receptor knockout mice. Endocr. Rev. 19, 225±268. Boutin, J. M., Jolicoeur, C., Okamura, H., Gagnon, J., Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane, J., and Kelly, P. A. (1988). Cloning and expression of the rat prolactin receptor, a member of the growth hormone/prolactin receptor gene family. Cell 53, 69±77. Boutin, J. M., Edery, M., Shirota, M., Jolicoeur, C., Lesueur, L., Ali, S., Gould, D., Djiane, J., and Kelly, P. A. (1989). Identification of a cDNA encoding a long form of prolactin receptor in human hepatoma and breast cancer cells. Mol. Endocrinol. 3, 1455±1461. Bridges, R. S., Dibiase, R., Loundes, D. D., and Doherty, P. C. (1985). Prolactin stimulation of maternal behavior in female rats. Science 227, 782±784. Buck, K., Vanek, M., Groner, B., and Ball, R. K. (1992). Multiple forms of prolactin receptor messenger ribonucleic acid are specifically expressed and regulated in murine tissues and the mammary cell line HC11. Endocrinology 130, 1108± 1114. Buteau, H., Pezet, A., Ferrag, F., Perrot-Applanat, M., Kelly, P. A., and Edery, M. (1998). N-Glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting. Mol. Endocrinol. 12, 544±555.

Chang, W. P., Ye, Y., and Clevenger, C. V. (1998). Stoichiometric structure±function analysis of the prolactin receptor signaling domain by receptor chimeras. Mol. Cell Biol. 18, 896±905. Chida, D., Wakao, H., Yoshimura, A., and Miyajima, A. (1998). Transcriptional regulation of the beta-casein gene by cytokines: cross-talk between STAT5 and other signaling molecules. Mol. Endocrinol. 12, 1792±1806. Clarke, D. L., Arey, B. J., and Linzer, D. H. (1993). Prolactin receptor messenger ribonucleic acid expression in the ovary during rat estrous cycle. Endocrinology 133, 2594±2603. Clement-Lacroix, P., Ormandy, C., Lepescheux, L., Ammann, P., Damotte, D., Goffin, V., Bouchard, B., Amling, M., GaillardKelly, M., Binart, N., Baron, R., and Kelly, P. A. (1999). Osteoblasts are a new target for prolactin: analysis of bone formation in prolactin receptor knockout mice. Endocrinology 140, 96±105. Clevenger, C. V., and Medaglia, M. V. (1994). The protein tyrosine kinase p59fyn is associated with prolactin (PRL) receptor and is activated by PRL stimulation of T-lymphocytes. Mol. Endocrinol. 8, 674±681. Clevenger, C. V., Torigoe, T., and Reed, J. C. (1994). Prolactin induces rapid phosphorylation and activation of prolactin receptor-associated RAF-1 kinase in a T-cell line. J. Biol. Chem. 269, 5559±5565. Clevenger, C. V., Chang, W. P., Ngo, W., Pasha, T. M., Montone, K. T., and Tomaszewski, J. E. (1995). Expression of prolactin and prolactin receptor in human breast carcinoma. Am. J. Pathol. 146, 695±705. Clevenger, C. V., Freier, D. O., and Kline, J. B. (1998). Prolactin receptor signal transduction in cells of the immune system. J. Endocrinol. 157, 187±197. Cunningham, B. C., Bass, S., Fuh, G., and Wells, J. A. (1990). Zinc mediation of the binding of human growth hormone to the human prolactin receptor. Science 250, 1709±1712. Das, R., and Vonderhaar, B. K. (1996). Involvement of SHC, GRB2, SOS and RAS in prolactin signal transduction in mammary epithelial cells. Oncogene 13, 1139±1145. De Vos, A. M., Ultsch, M., and Kossiakoff, A. A. (1992). Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255, 306±312. Dusanter-Fourt, I., Muller, O., Ziemiecki, A., Mayeux, P., Drucker, B., Djiane, J., Wilks, A., Harper, A. G., Fischer, S., and Gisselbrecht, S. (1994). Identification of Jak protein tyrosine kinases as signaling molecules for prolactin, Functional analysis of prolactin receptor and prolactin-erythropoietin receptor chimera expressed in lymphoid cells. EMBO J. 13, 2583±2591. Erwin, R. A., Kirken, R. A., Malabarba, M. G., Farrar, W. L., and Rui, H. (1996). Prolactin activates ras via signaling proteins Shc, growth factor receptor bound 2 and Son of Sevenless. Endocrinology 136, 3512±3518. Fuh, G., and Wells, J. A. (1995). Prolactin receptor antagonists that inhibit the growth of breast cancer cell lines. J. Biol. Chem. 270, 13133±13137. Gao, J., Hughes, J. P., Auperin, B., Buteau, H., Edery, M., Zhuang, H., Wojchowski, D. M., and Horseman, N. D. (1996). Interactions among janus kinases and the prolactin (PRL) receptor in the regulation of a PRL response element. Mol. Endocrinol. 10, 847±856. Gearing, D. P., Druck, T., Huebner, K., Overhauser, J., Gilbert, D. J., Copeland, N. G., and Jenkins, N. A. (1993). The leukemia inhibitory factor receptor (LIFR) gene is located within a cluster of cytokine receptor loci on mouse chromosome 15 and human chromosome 5p12-p13. Genomics 18, 148±150.

Prolactin Receptor 1561 Gertler, A., Grosclaude, J., and Djiane, J. (1997). Interaction of lactogenic hormones with prolactin receptors. Ann. N.Y. Acad. Sci. 839, 177±181. Ginsburg, E., and Vonderhaar, B. K. (1995). Prolactin synthesis and secretion by human breast cancer cells. Cancer Res. 55, 2591±2595. Goffin, V., Kinet, S., Ferrag, F., Binart, N., Martial, J. A., and Kelly, P. A. (1996a). Antagonistic properties of human prolactin analogs that show paradoxical agonistic activity in the Nb2 bioassay. J. Biol. Chem. 271, 16573±16579. Goffin, V., Shiverick, K. T., Kelly, P. A., and Martial, J. A. (1996b). Sequence±function relationships within the expanding family of prolactin, growth hormone, placental lactogen and related proteins in mammals. Endocr. Rev. 17, 385±410. Gout, P. W., Beer, C. T., and Noble, R. L. (1980). Prolactinstimulated growth of cell cultures established from malignant Nb rat lymphomas. Cancer Res. 40, 2433±2436. Helman, D., Sandowski, Y., Cohen, Y., Matsumoto, A., Yoshimura, A., Merchav, S., and Gertler, A. (1998). Cytokine-inducible SH2 protein (CIS3) and JAK2 binding protein (JAB) abolish prolactin receptor-mediated STAT5 signaling. FEBS Lett. 441, 287±291. Hennighausen, L., Robinson, G. W., Wagner, K. U., and Liu, W. (1997). Prolactin signaling in mammary gland development. J. Biol. Chem. 272, 7567±7569. Horseman, N. D., and Yu-Lee, L. Y. (1994). Transcriptional regulation by the helix bundle peptide hormones: growth hormone, prolactin, and hematopoietic cytokines. Endocr. Rev. 15, 627± 649. Horseman, N. D., Zhao, W., Montecino-Rodriguez, E., Tanaka, M., Nakashima, K., Engle, S. J., Smith, F., Markoff, E., and Dorshkind, K. (1997). Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene. EMBO J. 16, 6926±6935. Hu, Z. Z., Zhuang, L., Meng, J., and Dufau, M. L. (1998). Transcriptional regulation of the generic promoter III of the rat prolactin receptor gene by C/EBPbeta and Sp1. J. Biol. Chem. 273, 26225±26235. Ihle, J. N. (1996). STATs: signal transducers and activators of transcription. Cell 84, 331±334. Jabbour, H. N., Clarke, L. A., Boddy, S., Pezet, A., Edery, M., and Kelly, P. A. (1996). Cloning, sequencing and functional analysis of a truncated cDNA encoding red deer prolactin receptor: an alternative tyrosine residue mediates -casein promoter activation. Mol. Cell. Endocrinol. 123, 17±26. Jabbour, H. N., Critchley, H. O., and Boddy, S. C. (1998). Expression of functional prolactin receptors in nonpregnant human endometrium: Janus kinase-2, signal transducer and activator of transcription-1 (STAT1), and STAT5 proteins are phosphorylated after stimulation with prolactin. J. Clin. Endocrinol. Metab. 83, 2545±2553. Kelly, P. A., Djiane, J., Postel-Vinay, M. C., and Edery, M. (1991). The prolactin/growth hormone receptor family. Endocr. Rev. 12, 235±251. Kirken, R. A., Malabarba, M. G., Xu, J., Liu, X., Farrar, W. L., Hennighausen, L., Larner, A. C., Grimley, P. M., and Rui, H. (1997). Prolactin stimulates serine/tyrosine phosphorylation and formation of heterocomplexes of multiple STAT5 isoforms in Nb2 lymphocytes. J. Biol. Chem. 272, 14098±14103. Lange, C. A., Richer, J. K., Shen, T., and Horwitz, K. B. (1998). Convergence of progesterone and epidermal growth factor signaling in breast cancer. Potentiation of mitogen-activated protein kinase pathways. J. Biol. Chem. 273, 31308±31316. Lechner, J., Welte, T., Tomasi, J. K., Bruno, P., Cairns, C., Gustafsson, J., and Doppler, W. (1997). Promoter-dependent

synergy between glucocorticoid receptor and STAT5 in the activation of beta-casein gene transcription. J. Biol. Chem. 272, 20954±20960. Lesueur, L., Edery, M., Paly, J., Kelly, P. A., and Djiane, J. (1993). Roles of the extracellular and cytoplasmic domains of the prolactin receptor in signal transduction to milk protein genes. Mol. Endocrinol. 7, 1178±1184. Li, S., and Rosen, J. M. (1995). Nuclear factor I and mammary gland factor (STAT5) play a critical role in regulating rat whey acidic protein gene expression in transgenic mice. Mol. Cell. Biol. 15, 2063±2070. Lucas, B. K., Ormandy, C., Binart, N., Bridges, R. S., and Kelly, P. A. (1998). Null mutation of prolactin receptor gene produces a defect in maternal behavior. Endocrinology 139, 4102±4107. Luo, G., and Yu-Lee, L. Y. (1997). Transcriptional inhibition by STAT5: differential activities at growth-related vs differentiation-specific promoters. J. Biol. Chem. 272, 26841±26849. Mayr, S., Welte, T., Windegger, M., Lechner, J., May, P., Heinrich, P. C., Horn, F., and Doppler, W. (1998). Selective coupling of STAT factors to the mouse prolactin receptor. Eur. J. Biochem. 258, 784±793. Moldrup, A., Ormandy, C., Nagano, M., Murthy, K., Banville, D., Tronche, F., and Kelly, P. A. (1996). Differential promoter usage in prolactin receptor gene expression: hepatocyte nuclear factor 4 binds to and activates the promoter preferentially active in the liver. Mol. Endocrinol. 10, 661±671. Nagy, E., and Berczi, I. (1991). Hypophysectomized rats depend on residual prolactin for survival. Endocrinology 128, 2776±2784. Nicoll, C. S. (1974). In ``Handbook of Physiology: Endocrinology IV'' (ed. E. Knobil and W. Sawyer), Physiological actions of prolactin, pp. 253±292. Williams and Wilkins, Baltimore. Nicoll, C. S., and Bern, H. (1972). In ``Lactogenic Hormone'' (ed. G. Wolstenholme and J. Knight), On the actions of PRL among the vertebrates: is there a common denominator?, pp. 299±327. Churchill Livingstone, London. Nokelainen, P., Peltoketo, H., Vihko, R., and Vihko, P. (1998). Expression cloning of a novel estrogenic mouse 17 beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase (m17HSD7), previously described as a prolactin receptor-associated protein (PRAP) in rat. Mol. Endocrinol. 12, 1048±1059. Ormandy, C. J., Camus, A., Barra, J., Damotte, D., Lucas, B. K., Buteau, H., Edery, M., Brousse, N., Babinet, C., Binart, N., and Kelly, P. A. (1997a). Null mutation of the prolactin receptor gene produces multiple reproductive defects in the mouse. Genes Dev. 11, 167±178. Ormandy, C. J., Hall, R. E., Manning, D. L., Robertson, J. F., Blamey, R. W., Kelly, P. A., Nicholson, R. I., and Sutherland, R. L. (1997b). Coexpression and cross-regulation of the prolactin receptor and sex steroid hormone receptors in breast cancer. J. Clin. Endocrinol. Metab. 82, 3692±3699. Ormandy, C. J., Binart, N., Helloco, C., and Kelly, P. A. (1998). Mouse prolactin receptor gene: genomic organization reveals alternative promoter usage and generation of isoforms via alternative 30 -exon splicing. DNA Cell Biol. 17, 761±770. Perrot-Applanat, M., Gualillo, O., Pezet, A., Vincent, V., Edery, M., and Kelly, P. A. (1997). Dominant negative and cooperative effects of mutant forms of prolactin receptor. Mol. Endocrinol. 11, 1020±1032. Pezet, A., Ferrag, F., Kelly, P. A., and Edery, M. (1997). Tyrosine docking sites of the rat prolactin receptor required for association and activation of STAT5. J. Biol. Chem. 272, 25043±25050. Piccoletti, R., Maroni, P., Bendinelli, P., and Bernelli-Zazzera, A. (1994). Rapid stimulation of mitogen-activated protein kinase of rat liver by prolactin. Biochem. J. 303, 429±433.

1562 Vincent Goffin and Paul A. Kelly Posner, B. I., Kelly, P. A., Shiu, R. P., and Friesen, H. G. (1974). Studies of insulin, growth hormone and prolactin binding: tissue distribution, species variation and characterization. Endocrinology 95, 521±531. Postel-Vinay, M. C., Belair, L., Kayser, C., Kelly, P. A., and Djiane, J. (1991). Identification of prolactin and growth hormone binding proteins in milk. Proc. Natl Acad. Sci. USA 88, 6687±6690. Quijano, V. J. J., and Sheffield, L. G. (1998). Prolactin decreases epidermal growth factor receptor kinase activity via a phosphorylation-dependent mechanism. J. Biol. Chem. 273, 1200± 1207. Richer, J. K., Lange, C. A., Manning, N. G., Owen, G., Powell, R., and Horwitz, K. B. (1998). Convergence of progesterone with growth factor and cytokine signaling in breast cancer.Progesterone receptors regulate signal transducers and activators of transcription expression and activity. J. Biol. Chem. 273, 31317±31326. Riddle, O., Bates, R. W., and Dykshorn, S. W. (1933). The preparation, identification and assay of prolactin ± a hormone of the anterior pituitary. Am. J. Physiol. 105, 191±216. Rui, H., Kirken, R. A., and Farrar, W. L. (1994). Activation of receptor-associated tyrosine kinase JAK2 by prolactin. J. Biol. Chem. 269, 5364±5368. Shirota, M., Banville, D., Ali, S., Jolicoeur, C., Boutin, J. M., Edery, M., Djiane, J., and Kelly, P. A. (1990). Expression of two forms of prolactin receptor in rat ovary and liver. Mol. Endocrinol. 4, 1136±1143. Somers, W., Ultsch, M., De Vos, A. M., and Kossiakoff, A. A. (1994). The X-ray structure of the growth hormone-prolactin receptor complex. Nature 372, 478±481. Stocklin, E., Wissler, M., Gouilleux, F., and Groner, B. (1996). Functional interactions between STAT5 and the glucocorticoid receptor. Nature 383, 726±728. Stocklin, E., Wissler, M., Moriggl, R., and Groner, B. (1997). Specific DNA binding of STAT5, but not glucocorticoid receptor is required for their functional cooperation in the regulation of gene transcription. Mol. Cell. Biol. 11, 6708±6716. Stricker, P., and Grueter, R. (1928). Action du lobe anteÂrieur de l'hypophyse sur la monteÂe laiteuse. C.R. Soc. Biol. 99, 1978±1980. Touraine, P., Martini, J. F., Zafrani, B., Durand, J. C., Labaille, F., Malet, C., Nicolas, A., Trivin, C., PostelVinay, M. C., Kuttenn, F., and Kelly, P. A. (1998). Increased expression of prolactin receptor gene assessed by quantitative

polymerase chain reaction in human breast tumors versus normal breast tissues. J. Clin. Endocrinol. Metab. 83, 667±674. Wang, Y. F., O'Neal, K. D., and Yu-Lee, L. Y. (1997). Multiple prolactin receptor cytoplasmic residues and STAT1 mediate prolactin signaling to the IRF-1 promoter. Mol. Endocrinol. 11, 1353±1364. Wennbo, H., Gebre-Medhin, M., Gritli-Linde, A., Ohlsson, C., Isaksson, O. G., and Tornell, J. (1997). Activation of the prolactin receptor but not the growth hormone receptor is important for induction of mammary tumors in transgenic mice. J. Clin. Invest. 100, 2744±2751. Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998). Differential control of the phosphorylation state of proline-juxtaposed serine residues Ser725 of STAT5a and Ser730 of STAT5b in prolactin-sensitive cells. J. Biol. Chem. 273, 30218±30224. Yu-Lee, L. Y. (1997). Molecular actions of actions of prolactin in the immune system. Proc. Soc. Exp. Biol. Med. 215, 35±51. Yu-Lee, L. Y., Luo, G., Book, M. L., and Morris, S. M. (1998). Lactogenic hormone signal transduction. Biol. Reprod. 58, 295±301.

LICENSED PRODUCTS 1. Kelly, P.A., and Djiane, J. (1991) cDNA encoding human prolactin receptor. US patent number 4,992,378 2. Kelly, P.A., Edery, M., Prunet, M., and Sandra, O. (1994). cDNA encoding fish prolactin receptor. European patent number 94,10535

ACKNOWLEDGEMENTS The authors are grateful to Dr A.M. de Vos for providing the figure of the three-dimensional structure of the prolactin receptor extracellular domain (Figure 4). They also thank Dr N. Binart for helpful discussions and Dr A. Pezet for help with figures.