Discovery. Gerry Graham* and Rob Nibbs SUMMARY BACKGROUND

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1 D6 Gerry Graham* and Rob Nibbs Cancer Research Campaign Laboratories, The Beaton Institute for Cancer Research, Garscube Estate Switchback Road, Bearsdon, Glasgow G61 1BD, UK * corresponding author tel: , fax: , g.graham@beatson.gla.ac.uk DOI: /rwcy SUMMARY Members of the chemokine family of proinflammatory mediators interact with target cells by binding to members of the seven transmembrane spanning family of G protein-coupled receptors. We have been interested in identifying chemokine receptors involved in the regulation of hematopoietic stem cell proliferation and during these studies have cloned a novel chemokine receptor which is called D6. D6 is a chemokine-specific receptor but is highly promiscuous within this context and binds most members of this subfamily. Curiously, it does not seem to signal in the way that other chemokine receptors do and thus it has not yet been given a systematic name. D6 is expressed in the placenta and in the lung and liver but is detectable at low levels in most other tissues. Unlike many other chemokine receptors, it is not highly expressed in hematopoietic cells and thus its major domain of function may not be in this system. cysteines are juxtaposed. In addition to these two large families, there are two smaller subfamilies that have single members referred to as lymphotactin and fractalkine/neurotactin. All chemokines characterized thus far interact with cells through members of the seven transmembrane spanning heptahelical receptor family (Murphy, 1996) that typically support a calcium flux following ligand binding. A large number of chemokine receptors have been characterized so far and, in general, whilst they commonly display a marked promiscuity in terms of ligand binding, they are typically faithful to a single family. Thus, chemokine receptors will in general bind only chemokines and chemokine receptors will bind only chemokines. In this article we describe the identification and preliminary characterization of a novel chemokine receptor that displays atypical expression patterns and which is highly promiscuous but predictably faithful to chemokines. To date, we have been unable to demonstrate a signaling role for this novel receptor. BACKGROUND Chemokines are members of a large family of peptides that are involved in diverse processes such as regulation of inflammatory processes and control of cellular proliferation (Rollins, 1997). The chemokine family is defined on the basis of sequence homology but is more precisely defined by the presence of variations of a conserved cysteine motif. The two most populous chemokine subfamilies are the and subfamilies. Peptides in either of these families generally have four cysteine residues in the mature protein. In the chemokines the first two cysteines are separated by an amino acid of variable identity, whereas in the chemokines the first two Discovery We have been interested for some time in the role of chemokines, most notably MIP-1, as hematopoietic stem cell proliferation inhibitors (Graham et al., 1990; Graham, 1997; Graham and Nibbs, 1999). For a number of reasons, including the possible implication of MIP-1-inhibitory defects in the processes of leukemogenesis (Graham, 1997; Graham and Nibbs, 1999), we have been pursuing a number of strategies aimed at identifying cell surface receptors for MIP- 1. Our most successful strategy has involved degenerate genomic PCR using primers designed against conserved regions of the chemokine receptor family. Using this approach we identified a number of

2 2100 Gerry Graham and Rob Nibbs murine receptors for MIP-1, some of which have already been described but one of which, clone name D6, turned out to be novel (Nibbs et al., 1997a, 1997b). Alternative names Initially, given the ability of D6 to bind chemokines, this receptor was designated CCR9 in accordance with the existing systematic nomenclature system for chemokine receptors. Shortly after we were given the CCR9 designation for this molecule, a second group reported the cloning of this receptor (Bonini et al., 1997) and claimed a CCR10 nomenclature for it. The sequences for D6 presented in our publications and in that of Bonini et al. are essentially identical (Nibbs et al., 1997a, 1997b; Bonini et al., 1997), with the exception of three amino acids that may be accounted for by subtle allelic variations or alternatively by sequencing errors. The confusion in nomenclature has been further compounded by the fact that, despite extensive efforts, we have been unable to demonstrate a signaling or functional role for D6 in standard calcium flux or chemoattractant assays used to gauge the function of chemokine receptors. This lack of evidence of signaling capacity means that D6 is not entitled to a systematic nomenclature (signaling is a prerequisite for CCR designation) and thus it is now correctly referred to not as CCR9 or CCR10 but simply as D6. This therefore discriminates it from the recently described TECK receptor (GPR9-6) which does support calcium fluxing following ligand binding and which has been given a CCR9 nomenclature (Zaballos et al., 1999). We are currently examining the ability of D6 to signal through alternative pathways following ligand binding and hope in the near future to clarify the signaling potential for D6 and if appropriate to assign a systematic name to it. Structure D6 is a member of the seven transmembrane spanning family of receptors and as such is typical of other members of the chemokine receptor family. The murine D6 protein consists of 378 amino acids and the human protein of 384 amino acids. Both the murine and human proteins contain four cysteine residues that are conserved in other chemokine receptors and both also carry a potential N-linked glycosylation site at the N-terminus. Main activities and pathophysiological roles No data known on activities. This receptor is a coreceptor, with CD4, for the T-tropic HIV-1 isolate, UG21 and additionally displays coreceptor activity towards a number of other HIV-1 isolates (Choe et al., 1998). As such, D6 may have a limited role to play in the pathogenesis of HIV. In this pathological context, however, it is at best a weak player and the primary cellular coreceptors for HIV-1 remain CCR5 and CXCR4 (Clapham, 1997). GENE Accession numbers Murine cdna: Y12879 Rat cdna: U92803 Human cdna: Y12815, U94888 Additionally there are two human ESTs covering regions of D6 and accession numbers for these are R82383 and AI Sequence See Figure 1. Chromosome location and linkages Human D6 is located on chromosome 3 in a region coincident with the CC chemokine receptor locus at 3p p21.32 (Bonini et al., 1997). PROTEIN Accession numbers Rat D6: AAB61572 Murine D6: CAA73379 Human D6: CAA73346, AAB Sequence See Figure 2 for the human D6 protein sequence.

3 D Figure 1 Nucleotide sequence for the D6 gene. 1 GGATCCTCCA ACATGGCCGC CACTGCCTCT CCGCAGCCAC TCGCCACTGA GGATGCCGAT 61 TCTGAGAATA GCAGCTTCTA TTACTATGAC TACCTGGATG AAGTGGCCTT CATGCTCTGC 121 AGGAAGGATG CAGTGGTGTC CTTTGGCAAA GTCTTCCTCC CAGTCTTCTA TAGCCTGATT 181 TTTGTGTTGG GCCTCAGCGG GAACCTCCTT CTTCTCATGG TCTTGCTCCG TTACGTGCCT 241 CGCAGGCGGA TGGTTGAGAT CTATCTGCTG AATCTGGCCA TCTCCAACCT TCTGTTTCTG 301 GTGACACTGC CCTTCTGGGG CATCTCCGTG GCCTGGCATT GGGTCTTCGG GAGTTTCTTG 361 TGCAAGATGG TGAGCACTCT TTATACTATT AACTTTTACA GTGGCATCTT TTTCATTAGC 421 TGCATGAGCC TGGACAAGTA CCTGGAGATC GTTCATGCTC AGCCCTACCA CAGGCTGAGG 481 ACCCGGGCCA AGAGCCTGCT CCTTGCTACC ATAGTATGGG CTGTGTCCCT GGCCGTCTCC 541 ATCCCTGATA TGGTCTTTGT ACAGACACAT GAAAATCCCA AGGGTGTGTG GAACTGCCAC 601 GCAGATTTCG GCGGGCATGG GACCATTTGG AAGCTCTTCC TCCGCTTCCA GCAGAACCTC 661 CTAGGGTTTC TCCTTCCACT CCTTGCCATG ATCTTCTTCT ACTCCCGTAT TGGTTGTGTC 721 TTGGTGAGGC TGAGGCCCGC AGGCCAGGGC CGGGCTTTAA AAATAGCTGC AGCCTTGGTG 781 GTGGCCTTCT TCGTGCTATG GTTCCCATAC AATCTCACCT TGTTTCTGCA TACGCTGTTG 841 GACCTGCAAG TATTCGGGAA CTGTGAGGTC AGCCAGCATC TAGACTACGC ACTCCAGGTA 901 ACAGAGAGCA TCGCCTTCCT TCACTGCTGC TTTTCCCCCA TCCTGTATGC CTTCTCCAGT 961 CACCGCTTCC GCCAGTACCT GAAGGCTTTC CTGGCTGCCG TGCTTGGATG GCACCTGGCA 1021 CCTGGCACTG CCCAGGCCTC ATTATCCAGC TGTTCTGAGA GCAGCATACT TACTGCCCAA 1081 GAGGAAATGA CTGGCATGAA TGACCTTGGA GAGAGGCAGT CTGAGAACTA CCCTAACAAG 1141 GAGGATGTGG GGAATAAATC AGCCTGAGTG ACCGCGGCCG C Figure 2 Amino acid sequence for the D6 protein. 1 MAATASPQPL ATEDADSENS SFYYYDYLDE VAFMLCRKDA VVSFGKVFLP VFYSLIFVLG 61 LSGNLLLLMV LLRYVPRRRM VEIYLLNLAI SNLLFLVTLP FWGISVAWHW VFGSFLCKMV 121 STLYTINFYS GIFFISCMSL DKYLEIVHAQ PYHRLRTRAK SLLLATIVWA VSLAVSIPDM 181 VFVQTHENPK GVWNCHADFG GHGTIWKLFL RFQQNLLGFL LPLLAMIFFY SRIGCVLVRL 241 RPAGQGRALK IAAALVVAFF VLWFPYNLTL FLHTLLDLQV FGNCEVSQHL DYALQVTESI 301 AFLHCCFSPI LYAFSSHRFR QYLKAFLAAV LGWHLAPGTA QASLSSCSES SILTAQEEMT 361 GMNDLGERQS ENYPNKEDVG NKSA Description of protein The D6 protein describes a seven transmembrane spanning receptor with the characteristic extracellular N-terminus, intracellular C-terminus, and three extracellular loops. The murine D6 protein comprises 378 aminoacidsandthehumanprotein384aminoacidsand in addition both contain four conserved cysteine residues that are believed to be involved in the maintenance of chemokine receptor structure. The presumed extracellular N-terminus stretches from residue 1 to 46 and in common with other chemokine receptors is highly acidic, although the first 13 amino acids are predicted to form a hydrophobic domain that is not seen in the related chemokine receptors. Additionally, the N- terminus bears a consensus site for N-linked glycosylation (Asn/Ser/Ser) and additionally appears to be sulfated on tyrosine residues ± a decoration that is believed to be essential for its HIV coreceptor activity (Farzan et al., 1999). Whilst most chemokine receptors, indeed most seven transmembrane spanning G protein-coupled receptors, possess a conserved aspartate residue in the second transmembrane region (Savares and Fraser, 1992), this is altered to an asparagine in D6. An additional alteration in D6 compared to other chemokine receptors is that, whilst most signaling chemokine receptors possess a DRYLAIV motif on the second intracellular loop (Murphy, 1996) this is altered to DKYLEIV in D6. This alteration appears not to be random and is conserved in the rat, murine, and human receptors. It is this alteration in the conserved aspartate and the DRYLAIV motif that is likely to be the basis for the inability of D6 to flux calcium following ligand binding. Indeed, we have demonstrated that introduction of the DRYLAIV motif in D6 allows it to flux calcium following ligand binding, indicating that there is nothing radically different about the structure of D6 that precluded its support of calcium fluxing following ligand binding. The C-terminus of D6 bears many potential phosphorylation sites.

4 2102 Gerry Graham and Rob Nibbs Relevant homologies and species differences D6 is homologous to a number of members of the chemokine receptor family; however it is clearly quite divergent. Computerized packages assessing phylogenetic relationships between D6 and other chemokine receptors place it between the and chemokine receptor with the closest homology (40%) being seen with CCR4. Interestingly, comparison of the extracellular N-terminus of D6 shows it to be most similar to that of CCR1. Affinity for ligand(s) Whilst little is known as yet on the precise functional roles for this receptor, we have extensive data documenting the ligand-binding profiles of D6. D6 is a chemokine-specific receptor and will not bind members of the family, nor will it bind lymphotactin or fractalkine, the sole members of the other two smaller chemokine subfamilies. Despite this selectivity for chemokines, D6 is a very promiscuous receptor and will bind most chemokines, with some notable exceptions that are described below. This receptor is a very high-affinity MIP-1 receptor, with the murine receptor binding murine MIP-1 with a K d of 110 pm, making it the highest-affinity murine MIP-1 receptor described to date, and the human receptor-binding murine MIP-1 with a K d of 920 pm. In addition to MIP-1, human D6 will bind chemokines, as shown in Table 1. Thus, D6 is highly promiscuous and binds most members of the chemokine subfamily. Notable exceptions are C10 and I-309 and, perhaps most curiously, human MIP-1. Whilst murine D6 is the highestaffinity murine MIP-1 receptor described to date, it does not appear to bind the highly homologous human MIP-1. Equally, this ability to discriminate between murine and human MIP-1 is seen in human D6 which again binds murine MIP-1 with high affinity but binds human MIP-1 with a relatively lower affinity. We have recently attempted to examine the structural basis for this discriminatory ability and have demonstrated that a proline residue in position 2 of the chemokines is a prerequisite for high-affinity binding to human and murine D6 (Nibbs et al., 1999). Thus, all the ligands for this receptor and indeed, most chemokines, possess a proline 2 residue and thus bind well. It is important to note that a proline residue in position 2 of the mature protein is not sufficient in itself for D6 binding as the chemokine SDF-1, which has a proline 2 will not bind to D6. Additionally, a novel chemokine which we have identified (ESkine) also has a proline residue in position 2 but again will not bind to D6 (Baird et al., 1999). This however is the exception to the general rule. The relevance of the requirement for a proline residue in position 2 to the lack of binding of human MIP-1 to murine and human D6 lies in the fact that, Table 1 chemokines with which human D6 will bind Human Murine MIP-1 64 nm 920 pm MIP nm 755 pm RANTES 3.6 nm Not determined MCP nm 613 nm MCP pm Not determined MCP nm Not determined MCP nm Not determined MCP-5 Not determined 6.3 nm Eotaxin 46 nm 30 nm HCC nm Not determined C10 Not determined Undetectable I-309 Undetectable Not determined ESkine Not determined Undetectable

5 D unlike its murine counterpart, human MIP-1 carries a serine residue in position 2. There exists a natural nonallelic variant of human MIP-1 in the genome (Nakao et al., 1990) previously referred to as LD78 (the serine version of human MIP-1 is referred to as LD78 ) and we have demonstrated that this proline 2-bearing human MIP-1 isoform binds with high affinity to D6 and is therefore more like the murine protein in this and other contexts and may be the more representative human homolog of murine MIP- 1. We have proposed renaming LD78 and as MIP-1S and MIP-1P to highlight the importance of this subtle change (Nibbs et al., 1999). In terms of in vitro activities, as mentioned above, we have been unable to identify a role for D6. Unlike most other chemokine receptors, this receptor will not support either calcium fluxing or chemoattraction following ligand binding when expressed in heterologous cells. D6 therefore appears not to be a classical chemoattractant chemokine receptor and further work needs to be done to establish its biological relevance. Cell types and tissues expressing the receptor In the mouse, D6 is detectable on tissue blots at the level of northern blotting in liver, lung, spleen, and placenta. In addition, it is weakly expressed in murine hematopoietic cells and is detectable by PCR in murine dendritic cell lines (XS52 cells). The human mrna is very weakly expressed in most tissues examined; however it is robustly expressed in the placenta and is also easily detectable in the liver and lung. Expression in the human spleen is not detected at the level of northern blotting, perhaps reflecting the differences in cellular composition and function of the murine and human spleens. This receptor is not strongly expressed in human blood cells; however it can be detected by PCR in the primitive myeloerythroid cell line K562 and may indeed be present on the surface of these cells (Graham et al., 1993). Additionally, it is detectable in THP1 cells and in human umbilical cord blood cells. This expression pattern suggests that the primary domains of expression of D6 are not hematopoietic and that it may function in different contexts to those normally associated with chemokine receptors. This again may be reflected in the presumed alternative signaling pathways used by this receptor. Within the tissues mentioned above, we have few data on the specific cellular expression patterns of D6. We have recently obtained a monoclonal antibody to the human D6 protein and are in the process of using this to define cell types within the placenta, liver, lung, and other tissues expressing this receptor. Regulation of receptor expression No data known: however, preliminary analysis of a murine genomic clone reveals progesterone receptorbinding sites, cytokine response elements, camp response elements and glucocorticoid response elements. The relevance of these regulatory sites to transcriptional control remains to be determined. Release of soluble receptors No data known, although precedent from within the chemokine receptor family would suggest that soluble receptor release would be unlikely. SIGNAL TRANSDUCTION Cytoplasmic signaling cascades No data known, apart from the observations that, unlike most other chemokine receptors, D6 does not support calcium fluxing in heterologous transfectants following ligand binding. BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY Phenotypes of receptor knockouts and receptor overexpression mice Null mice have recently been generated and have no overt phenotypes. These mice are viable, healthy, and fertile. No data are yet available on D6 transgenic mice. THERAPEUTIC UTILITY Effect of treatment with soluble receptor domain No data known. Probably not relevant to chemokine receptors.