CCR8

CCR8 Monica Napolitano1,* and Angela Santoni 2 1

Laboratory of Vascular Pathology, Istituto Dermopatico dell'Immacolata-IRCCS, Via Monti di Creta 104, Rome, 0167, Italy 2 Department of Experimental Medicine and Pathology, University of Rome ``La Sapienza'', Via Regina Elena 324, Rome, 00161, Italy * corresponding author tel: 39-06-66462431, fax: 39-06-66462430, e-mail: [email protected] DOI: 10.1006/rwcy.2000.22008.

SUMMARY CCR8 is a CC chemokine receptor mostly homologous to CCR4 (44%). CCR8 mRNA has been detected in lymphoid tissues such as thymus, spleen, and lymph nodes. In particular, CCR8 message is expressed by CD4+ and CD4+CD8+ thymocytes, and by CD4+ and CD8+ peripheral blood T lymphocytes. This pattern of expression suggests a role for this receptor and its ligands in the differentiation, and activation/migration of T lymphocytes. CCR8 may also be considered as a molecule associated with the TH2 `program', being abundantly expressed by NK1.1+CD4+ cells and by TH2 cells that migrate in response to CCR8 ligands, thus suggesting an important role of I-309/CCR8 in allergic diseases. The main agonists of CCR8 are represented by the eukaryotic CC chemokine I-309 and by vMIP-I, an HHV-8-encoded protein, that induce both calcium mobilization and chemotaxis in cellular transfectants overexpressing CCR8. A role for this receptor in modulating HIV infection is indicated by the demonstration that several HIV and SIV isolates utilize CCR8 as a coreceptor for viral entry/fusion and by the inhibition of CCR8mediated HIV infection shown by I-309.

BACKGROUND Chemokines constitute a family of structurally related regulators of chemotaxis, adhesion, differentiation, and proliferation. They interact with a class of seven transmembrane-spanning receptors that are coupled to Gi or to a lesser extent to Gq protein, and whose

stimulation typically leads to intracellular calcium mobilization and cellular chemotaxis.

Discovery Human CCR8 The chemokine receptor superfamily shows structural motifs of G protein-coupled receptors, thereby representing a subfamily of this class of molecules. The degree of relatedness of these receptors in several regions of their primary sequence has prompted cloning og novel genes by low-stringency and degenerate PCR-based approaches. In particular, such methods were used to clone the human CCR8 gene from human cDNAs or genomic DNA, followed by hybridization of the amplified fragments to human genomic libraries (Napolitano et al., 1996; Samson et al., 1996; Zaballos et al., 1996; Tiffany et al., 1997). Like most chemokine receptor genes, the CCR8 gene is intronless. Murine CCR8 Low-stringency hybridization of the human CCR8 open reading frame (ORF) to 129/SV genomic libraries yielded several clones of the murine CCR8 gene containing the complete ORF and several kilobases of 50 and 30 regions (Goya et al., 1998; Zingoni et al., 1998). Human and murine CCR8 share 71% sequence homology at the protein level (see Figure 2).

Alternative names Human CCR8 is also known as TER1, ChemR1, CKR-L1, C-C CKR-8, GPR-CY6, and CMKBRL2.

2094 Monica Napolitano and Angela Santoni

Structure The predicted structure for CCR8 is that of a typical G protein-coupled receptor with seven hydrophobic moieties spanning the plasma membrane, an extracellular N-terminus, and an intracellular C-terminus. The tertiary CCR8 structure has not been solved.

Main activities and pathophysiological roles It has been shown that the human CCR8 receptor can function both as a coreceptor and as a fusion cofactor of HIV-1, HIV-2, and SIV strains in cell lines overexpressing the CCR8 cDNA (Rucker et al., 1997; Jinno et al., 1998; Simmons et al., 1998; Yi et al., 1998; Zhang et al., 1998; Albright et al., 1999; Chan et al., 1999; Dittmar et al., 1999; Singh et al., 1999). CCR8 is able to mediate the entry/fusion of the HIV1 strains 89.6, ADA, SF162, 2028, 2076, and the SIV strain mac316, the HIV-2 strain SBL6669, and a primary HIV type 1 group O isolate. Moreover, I-309 inhibits the infection of several HIV strains in a CCR8-dependent fashion (Horuk et al., 1998). Figure 1

CCR8 has recently been shown to be preferentially expressed by TH2 cells and clones that are able to respond to CCR8 ligands. This suggests a possible role for CCR8 and its ligands in allergic diseases. As the only reported receptor for I-309 is CCR8, it can be hypothesized that the ability of I-309 to inhibit the dexamethasone-induced apoptosis of the BW157 cell line (Van Snick et al., 1996), which abundantly expresses the CCR8 message (Goya et al., 1998; Zingoni et al., 1998), may indeed be CCR8-mediated and suggests that I-309/CCR8 may control cellular apoptosis and/or proliferation.

GENE

Accession numbers Human CCR8: U45983, U62556, Z79782, Y08456 Murine CCR8: Z98206, AF001277

Sequence See Figure 1.

Nucleotide sequence for the human CCR8 gene.

Sequence Human CCR8 CCGCCATGCCCGGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCGCCATGTTGGAAGGCTGGTCTTGAACCCCTGACCTCAGGTGATCTGC CCACCTTGGCCTCCCAAAGTGCTAGGATTACAGGCATGAGCCACAGCTCCCGGTCTATCATTTAACCTTAATTACATCTTTAAAGGCCCAAATA GTCTCACCCACTCCAAATAGTCACACCCACACCGGAGGTTGAGCACTTCAACACATGAATTTGGGGAGGACACAGTTCAGTCCATAACATCCCC CTAATTTTTAAAAAATAAAAATGTTTTTAAGGAGTGAATGTCTTTTATGTGTCTCTGTGACCAGGTCCCGCTGCCTTGATGGATTATACACTTG ACCTCAGTGTGACAACAGTGACCGACTACTACTACCCTGATATCTTCTCAAGCCCCTGTGATGCGGAACTTATTCAGACAAATGGCAAGTTGCT CCTTGCTGTCTTTTATTGCC TCCTGTTTGTATTCAGTCTTCTGGGAAACAGCCTGGTCATCCTGGTCCTTGTGGTCTGCAAGAAGCTGAGGAGCATCACAGATGTATACCTCTT GAACCTGGCCCTGTCTGACCTGCTTTTTGTCTTCTCCTTCCCCTTTCAGACCTACTATCTGCTGGACCAGTGGGTGTTTGGGACTGTAATGTGC AAAGTGGTGTCTGGCTTTTATTACATTGGCTTCTACAGCAGCATGTTTTTCATCACCCTCATGAGTGTGGACAGGTACCTGGCTGTTGTCCATG CCGTGTATGCCCTAAAGGTGAGGACGATCAGGATGGGCACAACGCTGTGCCTGGCAGTATGGCTAACCGCCATTATGGCTACCATCCCATTGCT AGTGTTTTACCAAGTGGCCTCTGAAGATGGTGTTCTACAGTGTTATTCATTTTACAATCAACAGACTTTGAAGTGGAAGATCTTCACCAACTTC AAAATGAACATTTTAGGCTTGTTGATCCCATTCACCATCTTTATGTTCTGCTACATTAAAATCCTGCACCAGCTGAAGAGGTGTCAAAACCACA ACAAGACCAAGGCCATCAGGTTGGTGCTCATTGTGGTCATTGCATCTTTACTTTTCTGGGTCCCATTCAACGTGGTTCTTTTCCTCACTTCCTT GCACAGTATGCACATCTTGGATGGATGTAGCATAAGCCAACAGCTGACTTATGCCACCCATGTCACAGAAATCATTTCCTTTACTCACTGCTGT GTGAACCCTGTTATCTATGCTTTTGTTGGGGAGAAGTTCAAGAAACACCTCTCAGAAATATTTCAGAAAAGTTGCAGCCAAATCTTCAACTACC TAGGAAGACAAATGCCTAGGGAGAGCTGTGAAAAGTCATCATCCTGCCAGCAGCACTCCTCCCGTTCCTCCAGCGTAGACTACATTTTGTGAGG ATCAATGAAGACTAAATATAAAAACATTTTCTTGAATGGCATGCTAGTAGCAGTGAGCAAAGGTGTGGGTGTGAAAGGTTTCCAAAAAAAGTTC AGCATGAAGGATGCCATATATGTTGTTGCCAACACTTGGAACACAATGACTAAAGACATAGTTGTGCATGCCTGGCACAACATCAAGCCTGTGA TTGTGTTTATTGATGATGTTGAACAAGTGGTAACTTTAAAGGATTCTGTATCCAAGTGAAAAAAAAAGATGTCTGACCTCCTTCATATGCAAAA ATATACCTTCAGAGACTGTCAGTAGGCTGGAAGAAGTGGATATTGAAGTTTTGACATCAATGATGAGGCTCCAGTTGTCTATGCATTGACTGAT GGTGAAATGGCTGGAGTGATTCTGAATCAAGGTGATTGTGATTATAGTGACAATGAAGATGATGCTATTAATACTGCATAAAAAGTGCCTGTAG ATGACATGGTGAAAATATTTGACAGGCTTATGGAAGGACTACAGCAGCACGCATTCATAACAGAACAAGAAATTATCTCAGCTTATAAAATCAA ACAGAGACTTCTAGACAAAAACCATTGTTGATGAGGCAGATGCCTCTAGAAGAGACGTTTAAAAGCCATCAAACACAATGCCTCATCTTCCCTG GAGGACCCACTTCCTGATCCCTCAACTGTGTCTGATGTTTCTTCTCATGTAAGAAATAAAAAATAAAAATAAAAAAATATATATTGGTATGTAA CTACAGGAAAAAAATAAAAAATATATAGTGGACAGTAACCTTTCAATCAAAACTCAGTATCATAAGTAGAGACTGAAAACTTGCCGTTATTGAT TGTTGTTATTAACAGCTGATACAGGTATTCTGCTGATGCTACTGCTGCCTAGTTACCATGAACACGTTTTTTCACTATTAATGGTGCGTCATAT TTTTTACTTTTAAGTACTTACGTGTGAGTAAGTGTAAGAAAATGATTGCTTATCAGTAGTATCAATGATTTACTCAATATCTGAATCACCTTGA TTCAGAACCATTTCAGCTGTTTCACCATCAGTCAATGAATAACAGCCTCATTGATGTCAAAAACTTCAATATCCACTTCTTTCAGCCTACTGTA GACTCTGGAAGTATACTTTTTGCATATGTAAGGAAGTCAGA

CCR8 2095

Chromosome location and linkages

Description of protein

The human CCR8 receptor was mapped on chromosome 3p21-23 by fluorescent in situ hybridization (FISH), and on chromosome 3p21-24 by performing PCR on radiation hybrids and on YAC clones (Napolitano et al., 1996; Samson et al., 1996; Tiffany et al., 1997). The murine gene was mapped by FISH to the telomeric f4 region of chromosome 9 (Zingoni et al., 1998).

The hCCR8 ORF encodes for a 355 amino acid protein that is predicted to belong to the chemokine receptor family. It does not possess any N-linked glycosylation sites in its N-terminal or extracellular regions, and shows four conserved cysteines that may be involved in disulfide bridges. One potential site of phosphorylation by PKC is present in the C-terminus of the molecule.

PROTEIN

Relevant homologies and species differences

Accession numbers Human CCR8: 1468979, 1668736, 1707884, 2465082 Murine CCR8: 2765843, 4049612

Sequence See Figure 2.

The human CCR8 protein is very similar to CC chemokine receptors (39±44% identity), and to CXCR (<37% identity). The murine ORF encodes for a 353 amino acid protein that shares 71% homology with CCR8, that is maximal in the transmembrane regions and minimal in the N- and C-termini (Figure 2).

Figure 2 Alignment between the human and murine CCR8 proteins. TM1 10 20 30 40 50 60 MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVFSLLGNSLVILVL ::::.. .:: .:::: ::.:..:::::.. .. : ::..::.:::..::::::::::: MDYTMEPNVT-MTDYY-PDFFTAPCDAEFLLRGSMLYLAILYCVLFVLGLLGNSLVILVL 10 20 30 40 50 TM2 TM3 70 80 90 100 110 120 VVCKKLRSITDVYLLNLALSDLLFVFSFPFQTYYLLDQWVFGTVMCKVVSGFYYIGFYSS : :::::::::.:::::: ::::::.:.::::. :::::::::.:::::::.:::::.:: VGCKKLRSITDIYLLNLAASDLLFVLSIPFQTHNLLDQWVFGTAMCKVVSGLYYIGFFSS 60 70 80 90 100 110 TM4 130 140 150 160 170 180 MFFITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWLTAIMATIPLLVFYQVASEDGV ::::::::::::::.::::::.:::: .::.: :.:::.:. :::::.::::::::::. MFFITLMSVDRYLAIVHAVYAIKVRTASVGTALSLTVWLAAVTATIPLMVFYQVASEDGM 120 130 140 150 160 170 TM5 190 200 210 220 230 240 LQCYSFYNQQTLKWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRLV :::..::..:.:.::.::.:..: ::::.::.:..:::..::.::. : :::.:.::.:: LQCFQFYEEQSLRWKLFTHFEINALGLLLPFAILLFCYVRILQQLRGCLNHNRTRAIKLV 180 190 200 210 220 230 TM6 TM7 250 260 270 280 290 300 LIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQQLTYATHVTEIISFTHCCVNPVIY : :::.::::::::::.:::::::..::::::. :.:. : ::::.::::::::::::: LTVVIVSLLFWVPFNVALFLTSLHDLHILDGCATRQRLALAIHVTEVISFTHCCVNPVIY 240 250 260 270 280 290 310 320 330 340 350 AFVGEKFKKHLSEIFQKSCSQIFNYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL ::.:::::::: ..::::::.:: ::::::: . :.. : .:.::.::..: :: AFIGEKFKKHLMDVFQKSCSHIFLYLGRQMPVGALERQLSSNQRSSHSSTLDDIL 300 310 320 330 340 350

2096 Monica Napolitano and Angela Santoni

Affinity for ligand(s) Human CCR8 Most studies aimed at identifying chemokine receptor ligands and at studying downstream signaling events have utilized cell lines reconstituted with single chemokine receptors. Tiffany et al. (1997) have generated cellular transfectants of the pre-B cell line 4DE4 expressing the human CCR8 cDNA and identified the CC chemokine I-309 as a CCR8 ligand as measured by the prompt calcium mobilization induced in these cell lines following stimulation, differently from several other chemokines tested. Similar results were obtained by Roos et al. (1997) in the pre-B cell line 300-19 and by Goya et al. (1998) that showed I-309-induced calcium mobilization and chemotaxis in 293/CCR8-transfected cell lines at a concentration of 10±100 nM. Bernardini et al. (1998) have identified two novel ligands, MIP-1 and TARC, of the CCR8 receptor. Such CC chemokines induce chemotaxis of the Jurkat/CCR8 but not of the mock cellular transfectants. These data are controversial in the light of several reports that showed neither binding to CCR8 nor calcium mobilization in response to such agonists in other cell types (Roos et al., 1997; Tiffany et al., 1997; Goya et al., 1998; Dairaghi et al., 1999; Endres et al., 1999; Garlisi et al., 1999). The chemotaxis induced in Jurkat/CCR8 transfectants is dependent on the overexpression of CCR8 in such cells, but it still remains to be demonstrated that ligand binding and calcium mobilization may occur. Therefore, at the moment, I-309 is the only `recognized' eukaryotic ligand for CCR8. Viruses, such as herpesviruses and poxviruses, often attempt to evade the immune system by producing molecules acquired from cellular genes. In particular, several chemokine or chemokine receptorlike viral proteins have been described and shown to be functional in eukaryotic hosts. It has been reported that the viral chemokine vMIP-II, encoded by Kaposi's sarcoma herpesvirus (KSHV ), is a ligand for CCR8 and is a chemoattractant for TH2 cells (Sozzani et al., 1998). More recent reports indicate that vMIP-I, also KSHVencoded, selectively engages CCR8 (IC50 ˆ 1.2ÿ2 nM) but not several other chemokine receptors (Dairaghi et al., 1999; Endres et al., 1999), acting as an agonist. Moreover, vMIP-II has been shown to behave as a vMIP-I and I-309 antagonist in cellular transfectants (Dairaghi et al., 1999; Luttichau et al., 2000) but is a broader-spectrum antagonist. An antagonistic effect has also been shown by the viral product of molluscum contagiosum virus ORF

MC148R, also known as vMCC-1 (Dairaghi et al., 1999), considered to act as an inhibitor of several chemokines (Damon et al., 1998), and recently demonstrated to bind selectively to CCR8 and block CCR8 function but not several other chemokine receptors' activities (Luttichau et al., 2000). Murine CCR8 Goya et al. (1998) reported that I-309 and TCA3, its murine homolog, induce calcium mobilization of 293/mCCR8 cellular transfectants at 100 nM and that TCA3 binds to mCCR8 transfectants with a Kd of 2 nM.

Cell types and tissues expressing the receptor Human CCR8 The human CCR8 gene shows a restricted mRNA expression in lymphoid tissues and cell lines. It is abundantly expressed by the thymus and at low levels by spleen, lymph nodes, CD4+ and CD8+ and IL-2treated T lymphocytes, adherent and LPS-treated monocytes, and, at very low levels, by CD19+ B cells and PMNs (Napolitano et al., 1996; Samson et al., 1996; Zaballos et al., 1996; Roos et al., 1997; Rucker et al., 1997; Tiffany et al., 1997). CCR8 expression in cell lines ranged between high and low levels in MOLT4 (immature T), HUT78 (CD4+ mature T), NK3.3 (NK-like) and Jurkat cells, while virtually absent in other cell lines, such as K562, U-937, RPMI 8866, YT-5, NKL, Jurkat, HL60, THP-1, MEG-01, RAJI, KG1-A, HEL 92.1.7, and JM-1. (Two different groups reported either CCR8 expression or no expression in the Jurkat cell line as measured by northern blot analysis, probably due to cellular variants.) It has been reported that the CCR8 message is abundantly expressed in human TH2 cell lines and clones while poorly expressed in TH1 cells (Zingoni et al., 1998) and upregulated following TCR and CD28 stimulation similarly to the CCR4 gene (D'Ambrosio et al., 1998). Moreover, TH2 CD4+ cells express more abundant levels of CCR8 message than TH2 CD8+ cells (Sozzani et al., 1998). The CCR8 mRNA is detectable as a single 4 kb transcript when northern blot analysis is performed. Murine CCR8 Murine CCR8 is abundantly expressed in the thymus and, at lower levels, in the spleen, lymph nodes, and lungs with multiple transcripts (about 2, 3, and 4 kb) (Goya et al., 1998; Zingoni et al., 1998). Among

CCR8 2097 different thymocyte subpopulations, CCR8 is expressed by single-positive CD4+ cells and, faintly, by CD4+CD8+ cells. Moreover, the CCR8 message is present in both cortical and medullary thymocytes as detected by in situ hybridization. Both activated mouse TH2 polarized cells and NK1.1+CD4+ cells abundantly express CCR8 message, while polarized TH1 cells do not (Zingoni et al., 1998). Among the cell lines tested, the thymoma cell line BW157 shows extremely high levels of CCR8 mRNAs, as well as the T lymphomas EL4 and S49. In contrast, the RW246.7, BAF3, P815, YAC, and WEHI.7 cell lines show no expression of the CCR8 message (Goya et al., 1998; Zingoni et al., 1998).

Regulation of receptor expression While resting T cells express very low levels of CCR8 message, this receptor, as mentioned above, is very abundantly expressed by TH2 cells and is upregulated following TCR and CD28 engagement (D'Ambrosio et al., 1998) and TCR and IL-2 treatment of polyclonal T cell lines (Sallusto et al., 1999).

SIGNAL TRANSDUCTION The presence of several serine and threonine residues in the receptor C-terminus suggests that CCR8 signaling, like other seven transmembrane spanning receptors, may be regulated through the phosphorylation of specific residues present in the C-terminus by serine/threonine kinases, whose activity is generally known to induce receptor desensitization. No data are yet available.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY A common event induced by the interaction of specific chemokine ligands with their cognate receptors is the chemotaxis of target cells. I-309 (Roos et al., 1997; Tiffany et al., 1997; Bernardini et al., 1998; Goya et al., 1998) and vMIP-II (Sozzani et al., 1998) induce chemotaxis of CCR8-transfected cell lines, and of TH2 cells (D'Ambrosio et al., 1998; Sozzani et al., 1998; Zingoni et al., 1998), while vMIP-I induces a chemotactic response in rat Y3 cells (Endres et al., 1999). The chemotactic activity shown by TARC and

MIP-1 in the Jurkat cell line (Bernardini et al., 1998) is still not conclusive for the attribution of such molecules as bona fide ligands of CCR8 as in other cell lines they showed no activity on CCR8 transfectants.

THERAPEUTIC UTILITY I-309 may potentially be useful in the control of HIV infection due to its inhibitory activity towards CCR8dependent HIV infection, although it is considered to be a `minor' coreceptor. As the I-309/CCR8 ligand/receptor pair associate with a TH2 phenotype, being involved in the activation/migration of this type of T cell, it may play a role in allergic diseases. Reagents such as neutralizing antibodies and CCR8 antagonists may therefore prove useful in an attempt to block CCR8 activity where it may contribute to pathological conditions. At present, both vMIP-II and vMCC-I have been reported to act as CCR8 antagonists (Dairaghi et al., 1999; Luttichau et al., 2000), even though vMIP-II activity may be altered in N-terminal sequence variants (Boshoff et al., 1997; Kledal et al., 1997).

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ACKNOWLEDGEMENTS We are grateful to Dr A. Zingoni, Dr G. Bernardini, and Dr G. Spinetti, and to Dr A. Zlotnik's and Dr A. Mantovani's laboratories for their important contributions to our studies.