CCR2

CCR2 Jose Miguel Rodriguez-Frade, Mario Mellado and Carlos Martinez-A.* Department of Immunology and Oncology, Centro Nacional de BiotecnologõÂ a, CSIC, Campus de Cantoblanco, Madrid, 28049, Spain * corresponding author tel: 34-915854559, fax: 34-913720493, e-mail: [email protected] DOI: 10.1006/rwcy.2000.22002.

SUMMARY The name CCR2 refers to two alternatively spliced chemokine receptors: CCR2A and CCR2B. Although first identified as the specific, high-affinity receptor for MCP-1 present in monocytic cell lines, other chemokines have been shown to elicit responses through CCR2. CCR2 is expressed in monocytes, macrophages, T and B lymphocytes, natural killer (NK) cells, dendritic cells (DC), and in nonlymphoid cells and tissues. As one of the first chemokine receptors to be cloned and characterized, it has been studied extensively. Its implication in several pathologies, its structure±activity relationship, the signaling pathways activated, as well as its regulation have been analyzed in detail and it has served as a model for the study of other chemokine receptors.

BACKGROUND

Discovery The presence of this receptor was inferred from the robust and specific responses of monocytes to MCP-1, which could not be accounted for by known chemokine receptors such as CCR1, CXCR1, or CXCR2. It was first cloned from total RNA of a monocytic cell line (Mono Mac 6) by Charo et al. (1994), using degenerate primers corresponding to sequences of the second and third transmembrane domains of CCR1, CXCR1, and CXCR2 receptors, and PCR with Mono Mac 6 cDNA. CCR2 exists in two forms, CCR2A and CCR2B, which represent alternately spliced variants of a single gene. Shortly after the first description, Yamagami et al. (1994) cloned a functional MCP-1 receptor from THP-1 cells using similar

strategies, corresponding to the CCR2B variant. Equivalent CCR2 receptors have been described in mice (Boring et al., 1996; Kurihara and Bravo, 1996) and rats (Jiang et al., 1998).

Alternative names In addition to the accepted term CCR2, references to this receptor can be found in the literature as MCP1R (for MCP-1 receptor), CC-CKR2, and multiple variations on these nomenclatures. It is worth noting that, in most cases, references made to CCR2 usually correspond to the CCR2B variant, since the CCR2A variant is poorly expressed on cell surface.

Structure Like the rest of the chemokine receptors, both forms of the CCR2 are members of family 1 of the G protein-coupled receptors. CCR2 consists of seven membrane-spanning regions, which are linked by three extracellular and three intracellular loops, an extracellular N-terminal region and intracellular Cterminal region. The extracellular regions are believed to participate in ligand binding, whereas the intracellular regions act in concert to transduce biological signals. Of particular structural interest is the presence of extracellular disulfide bonds, required for maintaining the structural integrity necessary for ligand-binding and receptor activation. As is the case with most chemokine receptors, there are several potential N-glycosylation sites in the N-terminal region. The conserved DRYLAIVHA motif in the second intracellular loop (amino acids 137±145) has an important role in signal transduction; a nonfunctional CCR2B receptor has been described, CCR2BY139F,

2042 Jose Miguel Rodriguez-Frade, Mario Mellado and Carlos Martinez-A. in which tyrosine 139 is replaced by phenylalanine (Mellado et al., 1998). The two spliced forms of the CCR2 differ at the Cterminal end, resulting in a shorter 2B tail with a completely different amino acid sequence. Several putative Ser/Thr phosphorylation sites, involved in desensitization and internalization of the receptor, are present in this C-terminal region.

Main activities and pathophysiological roles The principal function of CCR2 is considered to be mediating migration of different cell types in response to members of the monocyte chemoattractant proteins (MCP). Among the cells known to respond to a chemotactic stimulus through this receptor are monocytes, macrophages, basophils, dendritic cells, NK cells, and T and B lymphocytes. The response varies depending on the cell differentiation status and its degree of activation, however, which is reflected by the CCR2 expression on the surface of the cells. CCR2 mediates leukocyte adhesion and monocyte extravasation, and has been implicated in monocyte differentiation and homing of recently activated T cells. The pathophysiological effects of CCR2 derive mainly from this function as a mediator of cell migration. It is well established that CCR2 has a major role in inflammatory diseases, including asthma, chronic inflammation, experimental encephalomyelitis, and atherosclerosis (Baggiolini, 1998; Gonzalo et al., 1998; Lukacs et al., 1999). In fact, CCR2, together with CCR3, may extensively support the recruitment and/or activation of the different populations found within the asthmatic airways. Although not yet well documented, it may be involved in those pathologies in which its principal ligand, MCP-1, plays a role, including sepsis, tumor rejection, CNS trauma, multiple sclerosis, bacterial and viral meningitis, psoriasis, and glomerulonephritis (Luster, 1998; Rollins, 1998). CCR2 is not one of the main coreceptors for HIV-1 strains; in fact, it appears to act as a coreceptor only for certain SIV-1 strains and dual-tropic (X4/R5) strains as 89.6. Population studies nevertheless indicate the influence of a mutation in the CCR2 receptor, CCR2V64I, in AIDS progression (Smith et al., 1997). Individuals bearing this mutant CCR2, which is as functional as the wild-type receptor, show delayed HIV-1 infection through R5 and X4 strains. This has been linked to mutations in the promoter region of the CCR5 gene (Berger et al., 1999) and to the fact that this receptor forms heterodimers with both CXCR4 and CCR5 (Mellado et al., 1999), which in turn block HIV-1 infection.

GENE

Accession numbers EMBL: UO3882, UO3905, D29984, U80924, U95626

Sequence See Figure 1.

Chromosome location and linkages The gene (CMKBR2) (Figure 2) is clustered within 285 kb of chromosome 3p21 including CCR1 and CCR3. It comprises approximately 7 kb and consists of three exons and multiple polyadenylation sites. The first exon contains the 50 UTR; the second exon contains the entire ORF of CCR2B, and the third exon contains the carboxy-tail and 30 UTR of CCR2A. Strong consensus sequences for canonical 50 donor and 30 acceptor splice sites are found at all proposed intron/exon splice junctions (Wong et al., 1997).

PROTEIN

Accession numbers SwissProt: Human: P41597 Mouse: P51683 Rat: O55193

Sequence See Figure 3. Human: Length: 374 (CCR2A) or 360 amino acids (CCR2B). Molecular weight: 41,914 Da (Charo et al., 1994; Yamagami et al., 1994) Mouse: Length: 355 amino acids. Molecular weight: 40,901 Da (Kurihara and Bravo, 1996; Boring et al., 1997) Rat: Length: 373 amino acids. Molecular weight: 42,763 Da (Jiang et al., 1998)

Description of protein The protein consists of seven hydrophobic transmembrane domains linked by successive intracellular and

CCR2 2043 Figure 1 GGATTGAACA CGGTTTATCA TACGGTGCTC CTCTACTCGC ATAAACTGCA GATCTGCTTT TTTGGGAATG ATCTTCTTCA GCTTTAAAAG GCTGTGTTTG TATGTCTGTG ATTTTGGGGC ACCCTGCTTC ATCATGATTG TTCCAGGAAT CAGGTGACAG GTTGGGGAGA CAAAAACCAG CAAGGACTCC CTTCAGGACA CGTCTGGCTT GGAAGGCTGA TCCAGTTCCT AAAATTAAAG CATGTCAAAC ATTTTGAGCA AGGAGTTGGA ATGATGTCGT CTCTCAGGCT GTTTAATCAC CTAATTTGCC ATTGGTAAAG CGAGCCAAGT AAACACTGGG TCTACTTTCA TTTCACCTTC TGAAATGTAA TGTTGATAAA

Figure 2 gene.

Nucleotide sequence for the CCR2 gene (2232 bp).

AGGACGCATT GAAATACCAA CCTGTCATAA TGGTGTTCAT AAAAGCTGAA TTCTTATTAC CAATGTGCAA TCATCCTCCT CCAGGACGGT CTTCTGTCCC GCCCTTATTT TGGTCCTGCC GGTGTCGAAA TTTACTTTCT TCTTCGGCCT AGACTCTTGG AGTTCAGAAG TGTGTGGAGG TCGATGGTCG AAGAAGGAGC CACAGATGTG GAGGAGAGAG CATTTTTGAA CTGAAAACTG GTGAAAATGC GGTGGTATGT AGTGTGTGAT TTGAATCACA TGCTGCCAAA ATTCGAGTGT AGTGGGAACT AATGGAAGGT TAAGAATGTT CTTCTAGAAC GGCCACATGG ATATATTTGT ATACTGTTTT AG 2232

TCCCCAGTAC CGAGAGCGGT ATTTGACGTG CTTTGGTTTT GTGCTTGACT TCTCCCATTG ATTATTCACA GACAATCGAT CACCTTTGGG AGGAATCATC TCCACGAGGA GCTGCTCATC CGAGAAGAAG CTTCTGGACT GAGTAACTGT GATGACTCAC CCTTTTTCAC TCCAGGAGTG TGGAAAAGGA CTAGAGACAG TGATTCACAG ACTCCAGCTG TACAGGCATA CAACTTGTAA TGTATTAGTC TTGGGAGACT CTGTGGGCAC GTATACGCTC AGCCTTTTGT TTCAGTGCTT CCTAAATCAA GGAGAAGCTC CTTATGTTGC CAGGCAACTT CTAAAGAAGG ATGATCCTAA TAACAACTAT

Structural organization of the CCR2

extracellular loops, an extracellular N-terminus, and a cytoplasmic C-terminal tail. The conserved cysteine residues form disulfide bonds that link the extracellular loops and are required for maintaining the structural integrity necessary for ligand-binding and receptor activation. The extracellular regions also contain putative Nlinked glycosylation sites. The extracellular regions are involved in ligand-binding. Using chimeric CCR2/ CCR1 or CCR2/CCR5 receptors (Samson et al., 1997), it has been shown that the N-terminal region is

ATCCACAACA GAAGAAGTCA AAGCAAATTG GTGGGCAACA GACATTTACC TGGGCTCACT GGGCTGTATC AGATACCTGG GTGGTGACAA TTTACTAAAT TGGAATAATT ATGGTCATCT AGGCATAGGG CCCTATAACA GAAAGCACCA TGCTGCATCA ATAGCTCTTG AGACCAGGAA AAGTCAATTG AAATGACAGA TGTGAATCTT GGTTGGAAAA GAGTTCAGAC ATGTGGTAAA ACAGAGATAA GCTGAGTCAA ATTAGCCTAT CATCGCTGTC GTTTTGTTTT CGCAGATGTC ATTGGCTTCT CCTGAAGTAA CCAGTGTGTT GGGAACTAGA TTTCAGAAAG TGAATGCATA GATTTGGAAA

TGCTGTCCAC CCACCTTTTT GGGCCCAACT TGCTGGTCGT TGCTCAACCT CTGCTGCAAA ACATCGGTTA CTATTGTCCA GTGTGATCAC GCCAGAAAGA TCCACACAAT GCTACTCGGG CAGTGAGAGT TTGTCATTCT GTCAACTGGA ATCCCATCAT GCTGTAGGAT AGAATGTGAA GCAGAGCCCC TCTCTGCTTT GGTGTCTACG CAGTATTTTC TTTTTTTAAA GAGTTAGTTT TTCTAGCTTT CCCAATAGTT GTGCATGCAG ATCTCAGCTG GTATCATTAT CTTGATGCTC AATCAAAGCT GCAAAGACTT TCTGATCTGA CTCCCAAGCT AAGTGGGGAC AAATGTTAAG ATAAATCAAT

ATCTCGTTCT TGATTATGAT CCTGCCTCCG CCTCATCTTA GGCCATCTCT TGAGTGGGTC TTTTGGCGGA TGCTGTGTTT CTGGTTGGTG AGATTCTGTT AATGAGGAAC AATCCTGAAA CATCTTCACC CCTGAACACC CCAAGCCACG CTATGCCTTC TGCCCCACTC AGTGACTACA TGAAGCCAGT GGAAATCACA TTACCAGGCA CAAACTACCT TAGTAAAAAT GAGTTGCTAT GAGCTTAAGA GTTGATTGGC CATCTAAGTA GATCTCCATT GAAGTCATGC ATATTGTTCC TTTAAACCCT TCCTCTTAGT TGCAAGCAAG GGACTATGGC AGAGCAGAAC TTGATGGTGA GCTATAACTA

-60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220

required for high-affinity binding of MCP-1 (Monteclaro and Charo, 1996). Monoclonal antibodies raised against the N-terminal region of CCR2 (amino acids 24±38) and the third extracellular loop (amino acids 273±292), which have agonist and antagonist activities, indicate that the third loop is also involved in ligand-binding, whereas the N-terminus has a role in activating the receptor (Frade et al., 1997a). The importance of the extracellular loops has been dissected in detail, and determinants present in the first extracellular loop support high-affinity binding (Asn104 and Glu105), whereas charged amino acid residues in this loop (His100) are critical for ligand-induced activation of G proteins, without affecting ligand-binding affinity (Han et al., 1999a). All together, these data indicate that the extracellular surface of the receptor acts not only to bind the appropriate ligands, but also to elicit the conformational changes needed for receptor activation. This

2044 Jose Miguel Rodriguez-Frade, Mario Mellado and Carlos Martinez-A. Figure 3 Amino acid sequences for human (CCR2A and CCR2B), mouse, and rat CCR2. Human CCRA2 MLSTSRSRFI RNTNESGEEV LVFIFGFVGN MLVVLILINC SAANEWVFGN AMCKLFTGLY ARTVTFGVVT SVITWLVAVF FHTIMRNILG LVLPLLIMVI VYFLFWTPYN IVILLNTFQE NPIIYAFVGE KFRSLFHIAL LDGRGKGKSI GRAPEASLQD

TTFFDYDYGA KKLKCLTDIY HIGYFGGIFF ASVPGIIFTK CYSGILKTLL FFGLSNCEST GCRIAPLQKP KEGA

PCHKFDVKQI LLNLAISDLL IILLTIDRYL CQKEDSVYVC RCRNEKKRHR SQLDQATQVT VCGGPGVRPG

Human CCR2B MLSTSRSRFI RNTNESGEEV TTFFDYDYGA PCHKFDVKQI LVFIFGFVGN MLVVLILINC KKLKCLTDIY LLNLAISDLL SAANEWVFGN AMCKLFTGLY HIGYFGGIFF IILLTIDRYL ARTVTFGVVT SVITWLVAVF ASVPGIIFTK CQKEDSVYVC FHTIMRNILG LVLPLLIMVI CYSGILKTLL RCRNEKKRHR VYFLFWTPYN IVILLNTFQE FFGLSNCEST SQLDQATQVT NPIIYAFVGE KFRRYLSVFF RKHITKRFCK QCPVFYRETV TGEQEVSAGL ETADRVSSTF TPSTGEQEVS VGL Mouse MEISDFTEAY PTTTEFDYGD STPCQKTAVR AFGAGLLPPL GNVLMILVLM QHRRLQSMTS IYLFNLAVSD LVFLFTLPFW FGDAMCKLLS GFYYLGLYSE IFFIILLTID RYLAIVHAVF IITSIITWAL AILASMPALY FFKAQWEFTH RTCSPHFPYK LKLNLLGLIL PLLVMIICYA GIIRILLRRP SEKKVKAVRL LWTPYNLSVF VSAFQDVLFT NQCEQSKHLD LAMQVTEVIA YVFVGERFWK YLRQLFQRHV AIPLAKWLPF LSVDQLERTS LSAGF Rat MEDSNMLPQF KQIGAWILPP DLLFLLTLPF RYLAIVHAVF HTCGPYFPTI RHRAVRLIFA QVTETLGMTH

IHGILSTSHS LYSLVFIFGF WAHYAANEWV ALKARTVTFG WKNFQTIMRN IMIVYFLFWT CCVNPIIYAF

LFPRSIQELD VGNMLVIIIL FGNIMCKLFT VITSVVTWVV ILSLILPLLV PYNIVLFLTT VGEKFRRYLS

EGATTPYDYD ISCKKLKSMT GLYHIGYFGG AVFASLPGII MVICYSGILH FQEFLGMSNC IFFRKHIAKN

GAQLLPPLYS FLITLPLWAH AIVHAVFALK GPYFPRGWNN AVRVIFTIMI ETLGMTHCCI KNVKVTTQGL

GAQLLPPLYS FLITLPLWAH AIVHAVFALK GPYFPRGWNN AVRVIFTIMI ETLGMTHCCI DGVTSTNTPS

YSLVFIIGVV IDYKLKDDWI ALRARTVTLG SLKQWKRFQA IFAITLLFFL YTHCCVNPII SISPSTGEHE

DGEPCHKTSV DIYLFNLAIS IFFIILLTID FTKSEQEDDQ TLFRCRNEKK VVDMHLDQAM LCKQCPVFYR

N-terminal region is also involved in the activity of CCR2 as a coreceptor for some HIV-1 strains (Rucker et al., 1996). In accordance with their structural role, the seven transmembrane domains are the most highly conserved regions, not only among chemokine receptors, but in all GPCR, although several residues may be involved in ligand-binding or receptor activation. The intracellular regions are mainly involved in coupling the CCR2 to signaling molecules. A critical role in signaling through GPCR has been assigned to the DRY motif at the junction of the third TM and the second intracellular loop. In the case of CCR2, the Y139 in this motif is critical, as its mutation results in a loss-of-function CCR2 (Mellado et al., 1998). Study of chimeric CCR2/CCR1 receptors has identified the role of the third intracellular loop in G protein coupling (Arai and Charo, 1996). Other authors described differential coupling of CCR2A and CCR2B to G protein, thus assigning a

role for the C-terminal region in G protein coupling (Kuang et al., 1996). Several tyrosine residues susceptible to phosphorylation are present in the CCR2B, mainly in the C-terminus. In contrast, CCR2A, which has a completely different C-terminal region, lacks most of these tyrosine residues. Comparison of the activity of these variants would help elucidate the role of these tyrosines in receptor function. The cytoplasmic tails also contain serine and threonine residues susceptible to phosphorylation. The role of phosphorylation of these residues by GRK in receptor desensitization and internalization has been documented (Franci et al., 1996; Aragay et al., 1998). The differences between CCR2A and 2B must therefore influence receptor desensitization and cellular trafficking, although this question has not been fully addressed. The membrane-proximal region of the CCR2B C-terminus is involved in chemotaxis and signal transduction, although neither phosphorylation of Ser/Thr residues nor internalization is required for chemotaxis (Arai et al., 1997). Differences have also been shown in the trafficking of CCR2A and 2B to the membrane surface. Experiments using mutant receptors show that there are cytoplasmic retention signals in the C-terminal region (amino acids 316± 349); these signals result in clear surface expression of chimeras containing the CCR2B carboxy-tail, whereas mutant receptors with CCR2A C-terminal ends are found mainly in the cytoplasm (Wong et al., 1997). It has recently been suggested that chemokine receptors, including CCR2, can exist in multiple interconvertible states (Lee et al., 1999). The CCR2 dimerizes following ligand stimulation, although the receptor regions involved in this process remain to be elucidated. In fact, dimerization is the first step in the initiation of the signaling cascade not only of CCR2 (Rodriguez-Frade et al., 1999a), but also of CCR5 (Rodriguez-Frade et al., 1999b) and CXCR4 (VilaCoro et al., 1999). See Figure 4.

Relevant homologies and species differences CCR2 has the greatest amino acid similarity with CCR5 (71%), sharing nearly identical transmembrane domains with this receptor, as well as extraand intracellular loops. The main differences, which influence ligand specificity, are located in the Nterminal extracellular domains. Significant similarities are also found with CCR1 (55%) and CCR3 (50%). The murine CCR2 shows 80% similarity to the human CCR2B and 71% to human CCR2A.

CCR2 2045 Figure 4 CCR2 structure. Regions implicated in ligand binding, coupling of effector molecules and suggested targets for GRK and JAK are highlighted.

Only one CCR2 form has been described in mice which, in contrast to the human homologs, lacks potential N-glycosylation sites.

Affinity for ligand(s) MCP-1: 0.5  0.1 nM MCP-2: 3.0  1 nM MCP-3: 5.0 nM MCP-4: not determined (Gong et al., 1997)

Cell types and tissues expressing the receptor CCR2 expression was first established in monocytes. Since then, many other cell types have been shown to respond to MCP-1 and to express CCR2. In most cases, however, receptor expression is not constitutive and is detected only following stimulation or under inflammatory conditions. Constitutive CCR2 expression has been assessed in monocytes/macrophages and monocytic cell lines (Mono Mac 1 and 6, THP-1), although levels vary since its expression is regulated upon differentiation (Charo et al., 1994; Frade et al., 1997a; Fantuzzi et al.,

1999). Freshly isolated B cells from tonsils and peripheral blood also show constitutive CCR2 expression (Frade et al., 1997b). Peripheral blood T lymphocytes do not express CCR2 under resting conditions, although its expression is readily observed after activation and IL-2 stimulation (Loetscher et al., 1996). Both TH1 and TH2 cells express CCR2. Memory T cells (CD45RO‡) of the CD62L±CD26‡ subset express CCR2 (Rabin et al., 1999). Other cells known to express CCR2, at least under certain conditions, include neutrophils (only under inflammatory conditions (Johnston et al., 1999), elicited eosinophils (Lukacs et al., 1999), natural killer cells (Polentarutti et al., 1997; Sozzani et al., 1997), immature dendritic cells (Sallusto et al., 1998), and mast cells (Campbell et al., 1999). CCR2 mRNA expression has also been demonstrated in endothelial cells under inflammatory conditions (Weber et al., 1999a) and in lung fibroblasts derived from pulmonary granulomas after cytokine treatment (Hogaboam et al., 1999). Expression of CCR2 in astrocytes and cultured glia increased following induction of experimental allergic encephalomyelitis (Jiang et al., 1998). mRNA expression of CCR2 has been determined by northern blot in lung, kidney, heart, bone marrow, spleen, and thymus, but protein expression in the cell membrane is not well documented.

2046 Jose Miguel Rodriguez-Frade, Mario Mellado and Carlos Martinez-A.

Regulation of receptor expression The many functions of the chemokines, the redundancy observed among them in receptor usage, as well as their coordinated action described in several models, suggest that both chemokine and receptor expression must be tightly regulated. Several stimuli and pathophysiological conditions have been shown to up- or downregulate CCR2 expression. IL-2 upregulates CCR2 expression in monocytes (Sozzani et al., 1997), NK cells (Pollentaruti et al., 1997), and T lymphocytes (Loetscher et al., 1996). Other cytokines such as IL-4 and IL-15 upregulate CCR2 levels in different cell types (Hogaboam et al., 1999; Perera et al., 1999). Dexamethasone selectively upregulates CCR2 expression in monocytes by increasing mRNA half-life. Steroids devoid of glucocorticoid activity are inactive. This upregulation correlates with the chemotactic potential of the corresponding cells in response to CCR2 ligands. In cases of hypercholesterolemia, CCR2 upregulation is detected in monocytes, an effect mediated by plasma lipoproteins. LDL is responsible for CCR2 upregulation in both monocytes and monocytic cell lines, through LDL-derived cholesterol. In contrast, HDL reduces and even reverses the effect of LDL in monocytes (Han et al., 1998, 1999b). CCR2 is downregulated by LPS, making cells unresponsive to MCP-1 but not to other chemokines that activate other receptors, such as MIP-1. This downregulation has been observed in PBLs, monocytes, macrophages (Zhou et al., 1999), and NK cells (Sozzani et al., 1997), and is mediated through a decrease in mRNA half-life. Similar downregulation of CCR2 has been described for other microbial agents (Sica et al., 1997). TNF downregulates CCR2 in Mono Mac 6 and THP 1 monocytic cell lines (Weber et al., 1999b), an effect that is counteracted by OXLDL. TCR triggering with anti-CD3 antibodies downregulates CCR2 in memory/effector T cells (Sallusto et al., 1998). Diverse effects have been described for IFN in CCR2 regulation, including CCR2 upregulation in lung fibroblasts (Hogaboam et al., 1999), downregulation in monocytes (Penton-Rol et al., 1998), and no effect in neutrophils (Bonecchi et al., 1999), suggesting cell-type specific receptor regulation.

SIGNAL TRANSDUCTION Several biochemical events are stimulated by MCP-1, including inhibition of adenylate cyclase, activation of PLC, calcium flux, IP3 generation and

G protein-dependent mechanisms (Rollins, 1998; Pelchen-Matthews et al., 1999).

Associated or intrinsic kinases Although no intrinsic kinase activity has been documented, data in the literature support the association of several kinases with CCR2. The activation and association of members of the Janus kinase family to CCR2B have been described (Mellado et al., 1998). It is also known that MCP-1 promotes activation of members of the GRK Ser/Thr kinase family (Franci et al., 1996); these kinases associate to CCR2 to form a macromolecular complex that also involves -arrestin (Aragay et al., 1998).

Cytoplasmic signaling cascades The current view of CCR2 signaling indicates that this receptor couples to multiple signaling cascades. Following ligand stimulation, the first events are conformational changes in the receptor, leading to receptor dimerization (Rodriguez-Frade et al., 1999a). This dimerization allows JAK coupling to the CCR2, as well as receptor and JAK phosphorylation, which are needed for effective G protein coupling (Mellado et al., 1998). The JAK pathway not only initiates G protein signaling, but also activates the STAT family of transcription factors. These events are not exclusive to the CCR2, but also occur in other chemokine receptors such as CCR5 and CXCR4 (RodriguezFrade et al., 1999b; Vila-Coro et al., 1999), as well as GPCR from other families, including the angiotensin II receptor (McWhinney et al., 1997). Both JAK2 association and CCR2 tyrosine phosphorylation occur in the presence of pertussis toxin (PTX), indicating no Gi participation in this process. JAK2 dissociates from the receptor in a Gi-dependent manner, however, as it is blocked by PTX treatment. No MCP-1-induced, PTX-sensitive G protein-mediated physiological effects or Gi association to CCR2 are observed after treatment of Mono Mac 1 cells with the JAK inhibitor tyrphostin B42. This is not the case when cells are treated with other tyrphostins, indicating that inhibition of JAK2 kinase activity abolishes the association and activation of the G proteins responsible for this response. This, and the JAK2 association with the CCR2 receptor in PTX-treated cells, implies that the first event after MCP-1 binding to CCR2 in Mono Mac 1 cells is JAK2 kinase association. The conformational changes promoted by both ligand interaction and tyrosine kinase association induce Gi protein association to its binding site,

CCR2 2047 which is probably located in the second intracellular loop, as is the case for the IL-8 receptor (Damaj et al., 1996). Close examination of the intracellular domains of the different chemokine receptors reveals a highly conserved motif in the second intracellular loop (DRY[I/L]A[I/V]V[H/Q]A) (Murphy, 1994). This domain includes the residues critical for GPCR activation and is also present in other seven transmembrane receptors, such as the angiotensin II receptor, which is also associated with ligand-induced STAT activation (Marrero et al., 1995). When signaling was analyzed through a receptor in which the CCR2BY139 was replaced by phenylalanine, CCR2BY139F, MCP-1-triggered functional responses such as Ca2‡ mobilization, cell migration, and CCR2B tyrosine phosphorylation were not elicited, although the receptor binds MCP-1 as well as does the wild-type receptor. This lack of response is due to abolition of JAK2 kinase association to the receptor, and thus of the Gi protein (Mellado et al., 1998). Most of these responses induced through the CCR2 can be inhibited by PTX treatment, indicating that members of the Gi protein family are the primary transduction partners associated with the receptors (Murphy, 1994; Frade et al., 1997b). The physical association of Gi to the CCR2 in response to MCP-1 stimulus in a human monocytic cell line (Mono Mac 1) has been described (Aragay et al., 1998). Signaling studies of the CC chemokine receptors in transfected HEK-293 cells revealed potent, agonistdependent inhibition of adenylyl cyclase and mobilization of intracellular calcium, consistent with receptor coupling to Gi (Myers et al., 1995). In these studies, the calcium response was not completely blocked by PTX, suggesting that chemokine receptors may couple to multiple G proteins, such as Gi, Gq, or G16, depending on the chemokine receptor studied, and indicating that receptor±G protein pairings are cell type-specific (Al-Aoukaty et al., 1996; Arai and Charo, 1996). Following activation by the chemokine-triggered receptor, the heterotrimeric Gi protein dissociates into the subunit complex and the GTP-bound Gi subunit, which remains associated to the receptor, probably through interaction with one or more regions of the intracellular loops (Arai and Charo, 1996). Both events, receptor association and subunit dissociation, initiate independent intracellular signaling responses by acting on different effector molecules. G also acts as a docking protein, providing an interface for the GPCR, which would facilitate the interaction of GPCR in diverse signaling pathways. This is the case for the coupling of G protein-coupled receptor kinases (GRK; Wu et al., 1998), in which , but not heterotrimeric G protein or G, interacts with

the third intracellular loop of the M2±M3 muscarinic receptors. The association of with the activated CCR2 allows the formation of a ternary complex with GRK2, required for effective receptor phosphorylation. It has been shown that MCP-1 induces both release and GRK2 association to the activated CCR2, allowing the formation of a macromolecular complex that also includes arrestin (Aragay et al., 1998). The formation of such a complex is necessary to promote desensitization of the CCR2, and is the first step leading to its internalization. In fact, a CCR2 mutant in which Ser/Thr residues of the C-terminal region were replaced by alanines shows greatly reduced internalization (Arai et al., 1997). The role of this region in chemotaxis is not conclusive, as although this mutant CCR2 supports chemokine-induced chemotaxis, other observations implicate the C-terminal region of CCR2 (amino acids 316±328) in chemotaxis. Regarding the GRK2-mediated desensitization process, both the GRK2 catalytic activity as well as the Ser/Thr residues in the CCR2B C-terminal domain are critical. Coexpression of GRK2 and CCR2 blocks MCP-1-induced responses. When a CCR2 receptor mutant lacking Ser/Thr residues in the carboxyl tail was expressed, the MCP-1-induced signal was not inhibited by GRK2 coexpression (Franci et al., 1996). This critical role of GRK kinase in CCR2 deactivation was also demonstrated when the CCR2 receptor was coexpressed with a dominant negative GRK2 mutant (Kong et al., 1994); the cellular response to a second MCP-1 challenge was equivalent to the original response (Franci et al., 1996). The formation of phosphatidylinositol 3-kinase (PI-3 kinase) lipid products has also been observed after CCR2 activation, a process which is inhibited by PTX. MCP-1 activates at least two different PI-3 kinase isoforms, p85/p110 and C2. The biological consequences of this activation remain to be clarified, although PI-3 kinase activation has been implicated in a variety of cellular responses such as adhesion molecule upregulation (Shimizu and Hunt, 1996), superoxide release (Chung et al., 1994), and chemotaxis (Turner et al., 1995; Knall et al., 1997; Turner et al., 1998). PTX-dependent phospholipase C (PLC) activation through MCP-1-activated CCR2B and 2A has been described (Kuang et al., 1996). GPCR signaling is linked to the mitogen-activated protein kinase (MAPK) cascade. MCP-1 binding to CCR2B promotes rapid, transient activation of MAPK. This process appears to be PTX-insensitive and PKC-dependent. Although its role remains to be clarified, several authors include this pathway among those required for chemotaxis (Yen et al., 1997) (Figure 5).

2048 Jose Miguel Rodriguez-Frade, Mario Mellado and Carlos Martinez-A. Figure 5 Schematic representation of the signaling pathways activated through the CCR2. Ligand-binding to CCR2 induces conformational changes in the receptor, leading to its dimerization; this in turns disables JAK2 association and activation. JAK activation initiates the JAK/STAT pathway and enables efficient G protein coupling to CCR2. G proteins mediate signaling cascades through GRK and arrestins, leading to receptor desensitization and internalization. Other signaling molecules triggered through CCR2 include PI-3 kinase, PLC, and MAPK.

DOWNSTREAM GENE ACTIVATION

Transcription factors activated JAK2/STAT5 pathway activation by MCP-1 has been reported (Mellado et al., 1998), although other

members of the JAK/STAT pathway may also be activated. The consequences of this activation remain to be elucidated. The fact that other chemokines, such as RANTES, activate transcription factors such as JNK through MAPK-related pathways, suggests that CCR2 may mediate activation of similar factors.

CCR2 2049

Promoter regions involved 1. Transcription initiation sites 26 bp downstream from the TATA box. 2. Consensus AATAAA polyadenylation signal sequences upstream of the 30 exon 2 and 3 (Wong et al., 1997), consistent with the presence of two different CCR2 transcripts. 3. Oct-1 binding sequences 36 bp upstream TATA box, important for transcriptional activation of CCR2. 4. Tandem CAAT/enhancer-binding protein (C/ EBP) binding sequences at ‡50 and ‡77 within the 50 UTR, which are essential for transcriptional activation and tissue specificity of CCR2. 5. Typical mammalian promoter consensus elements TATAA (ÿ31 to ÿ27) and CCAAT (ÿ128 to ÿ124). It is interesting to note its different transcriptional activation compared with CCR5, although both receptors are supposed to share the same ancestral gene (Yamamoto et al., 1999).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors As for other chemokine receptors, one of the first consequences of CCR2 activation is a rapid, transient elevation of intracellular calcium that is PTXsensitive. In accordance with Gi pathway activation, inhibition of cAMP production after CCR2 activation has also been described (Monteclaro and Charo, 1997). Another classical effect seen after CCR2 activation is the accumulation of inositol phosphate, related to PLC activation. One of the most analyzed chemokine-related phenomena is chemotaxis. Chemotaxis is a key phenomenon both in cell movement and in the inflammatory response, and CCR2-mediated chemotaxis is welldocumented (Rollins, 1998). It is implicated in the polarization of lymphocytes during migration, such that the leading cell edge develops cytoplasmic extensions, whereas the posterior part of the cell forms an appendage, the uropod. Following stimulation by molecules such as MCP-1, RANTES, IL-8, IL-15 or IL-2, the CCR2 is redistributed and located at the

advancing front of migrating lymphocytes (Nieto et al., 1997). It has recently been demonstrated that NK cells contact target cells through the advancing front, a region in which CCR2 and CCR5 are concentrated; this suggests a role for these receptors during cytotoxic phenomena (Nieto et al., 1998).

Phenotypes of receptor knockouts and receptor overexpression mice While there are no references to mice overexpressing CCR2, several authors have developed CCR2-knockout mice. CCR2 deficiency has no observable effects on growth, development, or fertility. With respect to the immune response, the mice are not severely immunocompromised, with normal hematopoietic development including myeloid progenitors and development of lymphocyte subsets. The biological consequences nevertheless become clear when mice are challenged with proinflammatory stimuli. In vivo, it appears that CCR2 is highly specific for MCP-1, in accordance with the fact that MCP-1-knockout mice display a phenotype similar to that of the CCR2ÿ/ÿ mice (Rollins, 1996; Lu et al., 1998). Kuziel et al. (1997) reported that CCR2 is required for firm adhesion and diapedesis of leukocytes, which results in reduced macrophage accumulation at inflammation sites. Similar defects were observed in macrophages recovered from CCR2ÿ/ÿ mouse peritoneum after nonspecific inflammatory challenge (Kurihara et al., 1997), whereas neutrophils and eosinophils are unimpaired, indicating the selectivity of CCR2 in eliciting such responses. This decrease in macrophage recruitment, which is not cholesterolmediated, results in decreased lesion formation in CCR2ÿ/ÿ mice backcrossed with apoE null mice, which develop severe atherosclerosis (Boring et al., 1998). These defects compromise the macrophagedependent immunity to intracellular pathogens (Kurihara et al., 1997). Impaired monocyte and leukocyte migration has been observed in CCR2-knockout mice, as well as decreased IFN and IL-2 production following exposure to antigenic and nonantigenic challenge. It is not yet clear whether this decrease in IFN production is due to a direct effect of CCR2 on T cells, or to defects in CCR2-mediated trafficking to lymph nodes or spleen (Boring et al., 1997). Defects in macrophage recruitment and host defense, and enhanced myeloid progenitor cell cycle and apoptosis in bone marrow but not in spleen, have been described (Reid et al., 1999). It is interesting to note that other myelosuppressive chemokines, acting

2050 Jose Miguel Rodriguez-Frade, Mario Mellado and Carlos Martinez-A. through non-CCR2 receptors, do not have compensatory suppressive effects. An altered airway hyperreactivity response, with decreased histamine levels following challenge, has also been described in CCR2ÿ/ÿ mice (Campbell et al., 1999).

Human abnormalities A polymorphism has been reported in CCR2, in which Val64 is replaced by Ile, giving the CCR2V64I (Smith et al., 1997). This polymorphism occurs at an allele frequency of 10±25%, depending on the ethnic population. There is no evidence of functional difference between the CCR2V64I and the wild-type CCR2; however, this polymorphism is associated with a 2±4year delay in progression to AIDS in HIV-1-infected individuals. More recently, an increased incidence of this polymorphism has been described in children with insulin-dependent diabetes mellitus (Szalai et al., 1999), whereas the presence of the CCR2V64I allele confers a lower risk for development of sarcoidosis (Hizawa et al., 1999). Whether or not these effects are directly related to CCR2 function remains to be elucidated.

THERAPEUTIC UTILITY

Effects of inhibitors (antibodies) to receptors Antibodies against CCR2 have been developed by several groups (Frade et al., 1997b; Sallusto et al., 1998), some of which can block MCP-1-induced effects. This blockage results in reduced migration both in mouse models and in in vitro assays, as well as in reduced calcium mobilization and arachidonic acid release; some anti-CCR2 antibodies also neutralize HIV-1 strains. Modified MCP-1 with antagonist activity has also been described (Zhang et al., 1996).

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LICENSED PRODUCTS Antibodies against CCR2, both monoclonal and polyclonal, are available from several suppliers, including R&D Systems (614 McKinley Place, Minneapolis, MN 55413, USA, http://www.rndsystems.com) and Santa Cruz (2161 Delaware Ave., Santa Cruz, CA 95060, USA, http://www.scbt.com). Among these are antibodies suitable for flow cytometry analysis, western blot and immunohistochemistry. Some antibodies have been derived using peptides from the CCR2 C-terminal tail; although

they are only suitable for analysis of CCR2 in permeabilized or lysed cells, they offer the advantage of specific distinction between CCR2A and 2B variants. Note that some of the antibodies available may react differently with CCR2, depending on the cell line employed. The corresponding recombinant ligands of human, rat, and murine origin are available from Peprotech (5 Crescent Ave., Rocky Hill, NJ 08553-0275, USA, http://www.peprotech.com), Pharmingen (10975 Torreyana Road, San Diego, CA 92121-1111, USA, http://www.pharmingen.com), Genzyme (One Kendall Square, Cambridge, MA 02139, USA, http://www.genzyme.com), and R&D Systems (614 McKinley Place, Minneapolis, MN 55413, USA, http://www.rndsystems.com). Radiolabeled ligands are available from Amersham (http://www.apbiotech.com) and NEN (http://www.nenlifesci.com). A multi-probe ribonuclease protection assay for mRNA analysis of chemokines and chemokine receptors (including CCR2) is available from Pharmingen.