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Structure and Functional Expression of the Human Inflammatory Protein lc~/RANTES Receptor By Ji-Liang Gao,* Douglas B. Kuhns,~ H. Lee Tiffany,* David McDermott,* Xu Li,$ Uta Francke,$ and Philip M. Murphy*

From the *Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Heahh, Bethesda, Maryland 20892; *Clinical Services, Program Resources, Ina/DynCorp, National Cancer Institute, Frederick Research and Development Center, Frederick, Maryland 21072; and the SDepartment of Genetics and Howard Hughes Medical Institute, Stanford University Medical Center, Stanford, California 94305

Summary The ~ family is comprised of at least six distinct that regulate trafficking of phagocytes and in mammalian species; at least one of these, macrophage inflammatory protein lcr (MIP-loL), also regulates the growth of hematopoietic stem cells. We now show that MIP-loe and the related ~/chemokine, RANTES, induce transient alterations in intracellular Ca 2+ concentration in polymorphonuclear leukocytes that can be reciprocally and specifically desensitized, suggesting a common receptor. Moreover, we have now cloned both the cDNA and the gene for this receptor, functionally expressed the receptor in Xenopus oocytes, and mapped the gene to human chromosome 3p21. Transcripts for the receptor were found in mature and immature myeloid cells as well as B cells. The receptor is a member of the G protein-coupled receptor superfamily. It has "~33% amino acid identity with receptors for the oe chemokine, 8, and may be the human homologue of the product of US28, an open reading frame of human cytomegalovirus.

hemokines are structurally and functionally related 8-10- inflammatory protein la (MIP-lo01, MIP-1B, regulated on C kD peptides that are the products of distinct genes activation, normal T expressed and secreted (RANTES), clustered on human chromosomes 4 and 17. regu- monocyte chemoattractant protein 1 (MCP-1), MCP-2, late the traffcking and activation of lymphocytes and phago- MCP-3, and 1-309 (3). The biological properties of MIP-la, cytes of the mammalian . In addition, some RANTES, and MCP-1 have been the most characterized (Table are able to regulate the proliferative potential of hematopoi- 1). Members of this group attract and activate PMN, eo- etic progenitor ceils, endothelial ceils, and certain types of sinophils, monocytes, and lymphocytes with variable selec- transformed cells (reviewed in references 1-3). Thus, the tivity (7-11). In addition, MIP-loe has been shown to regu- chemokines may play an important role in host defense against late the proliferative capacity of myeioid progenitor cells infection, in the pathogenesis of chronic inflammatory dis- (12-15). orders, and in wound healing. To more precisely delineate the role of chemokines in the The chemokines can be divided into two groups, ot and inflammatory response and hematopoiesis, we wished to study 8, by the arrangement of the first two of four conserved cys- the structure and signal transduction properties of their teines. Members of the chemokine c~ group all possess a single receptors. Here we report the cloning of the cDNA and amino acid of variable identity interposed between the first the gene for the first chemokine ~ receptor, the MIP-lcr two cysteines. IL-8 is the best characterized member of this RANTES receptor. group. Two human IL-8 receptors have been identified that are 77% identical at the amino acid level (4, 5). IL-SR B can also be activated by the related c~ chemokines, GROc~ and neutrophil activating peptide 2 (5, 6). I Abbreviations used in this paper: MCP-1, monocyte chemoattractant The first two conserved cysteines of 3 chemokines are ad- protein 1; MIP-lc~, macrophage inflammatory protein la; RANTES, jacent. The human chemokine 3 family includes macrophage regulated on activation, normal T expressed and secreted.

1421 The Journal of Experimental Medicine Volume 177 May 1993 1421-1427 Materials and Methods eDNA and Genomic Library Screening. A 2-kb Hit60 neutro- The RNA and DNA isolation, Northern and Southern blot anal- phil cDNA library in h ZAP (Stratagene, La Jolla, CA) was screened ysis, and DNA sequencing were done exactly as described (16). with a 32p-labded oligonucleotide probe corresponding to nucleo- Intracelhlar Calcium Measurements in Blood Cells. PMN and tides 238-276 of the cDNA sequence of clone F3K, which en- PBMC were isolated from peripheral blood by Ficoll-Hypaque dis- codes the rabbit IL8K (18), exactly as described (5). DNA sequences continuous density gradient centrifugation. PMN were further were determined on both strands of the two longest hybridizing purified by dextran sedimentation and hypotonic lysis. PMN were clones, designated p4 and p7. A human genomic library in ~ FIX typically contaminated with '~5% eosinophils. PBMC were -020% (Stratagene) was screened exactly as described (19) with a 3zp. monocytes and 80% lymphocytes. Cells (107/ml) were washed labeled probe of p4 cDNA synthesized from random primers. with HBSS buffered with Hepes (10 mM, pH 7.4) and loaded with Restriction sites of positive clones were mapped and a 6.5-kb XbaI 2.5 pM Indo-1 AM (Molecular Probes, Eugene, OR) for 30 min fragment was sequenced. at 37~ in the dark. The cells were subsequently washed twice Calcium E.fftu.xAssay. cRNA was prepared exactly as previously with buffer and resuspended at 2 x 106 cells/ml. 3 ml of the cell described (5). The materials and methods used for the calcium dtlux suspension was placed in a continuously stirred, quartz cuvette main- assay were as described (5) with minor modifications. Defollicu- tained at 37~ (DeltaScan; Photon Technology International Inc., lated oocytes from Xenopus laevis were microinjected with RNA S. Brunswick, NJ). Fluorescence was monitored at K.~ = 358 nm samples 1-2 d after harvesting and were then incubated at 20-23~ and ~m m 402 nm (bound) and 486 nm (free), and the data are for 3-5 d. Oocytes were then incubated with 45Ca2+ (100/.LCi/ml; presented as the bound/free ratio. Data were collected every 200 Amersham Corp., Arlington Heights, IL) for 4 h. After 10 washes ms. The Rm~ and Rmin were empirically obtained by adding with medium, individual oocytes were stimulated with ligands. ionomycin (1 pM) and EGTA (10 mM), respectively. The dissocia- Data are presented as the mean _+ SEM of the percent of loaded tion constant of Indo-1 (Ka = 250 riM) was used to transform 4SCa2+ that was released in 20 rain by individual oocytes in re- the data to [CaZ+]i in the relationship [Ca2+] = Ka. [(K - Rmin) sponse to the stimulus. Recombinant human ligands were obtained + (Rm,~ - R)" (Sfe/Sb2)(17). as follows: MIP-lc~ (R & D Systems, Minneapolis, MN); I1_,8,

Table 1. The Human Chemokine ff Family of Proinflammatory Peptides

Percent identity to mature MIP-lc~ Chemokine* (sequence accession no.) Source* Targets/actions

MIP-la 100 (D90144) B & T lymphocytes Neutrophil and monocyte chemotaxis; ~11 Mast cells neutrophilSll and activation; stem Fibroblasts cell suppression; potentiation of GM-CFU Macrophages stimulation by GMCSF;S PGE-independent Monocytes endogenous pyrogenSII MIP-1B 67 (B30574) B & T lymphocytes Inhibits MIP-lcr action on stem cells; s Macrophages potentiates GM-CFU stimulation by GMCSF; s Monocytes inhibits macrophage activation by MIP-lc~S RANTES 46 (M21121) T lymphocytes Monocyte, granulocyte, and memory Platelets T (CD4+/CD45Ro +) chemotaxis 1-309 38 (M57506) T lymphocytes Mouocyte chemotaxis MCP-1 39 (M24545) Monocytes Monocyte chemotaxis and activation; Endothelial cells tumor suppression s Fibroblasts Keratinocytes MCP-2 40 (P80075) MG-63 osteosarcoma Monocyte chemotaxis cells MCP-3 34 (P80098) MG-63 osteosarcoma Monocyte chemotaxis cells

* Additional information regarding alternative names and sequence variants of human and murine B chemokines can be found in references 1-3. * The sources listed are based on protein purification and/or chemokine RNA distribution in normal human cell types with the exception of MCP-2 and MCP-3, for which information is currently available only for the transformed cell type from which they were purified. S Activities so far reported only for the murine homologue. It Activities so far reported only for native murine MIP-1, which is purified as a heterodimer of MIP-I~ and MIP-1/~. Human and routine forms of mature MIP-lc~ and MIP-lfl peptides are 74 and 77% identical in amino acid sequence, respectively.

1422 The Human Macrophage Inflammatory Protein lot/RANTES Receptor RANTES, and MCP-1 (Genzyme, Cambridge, MA); NAP-2 situ hybridizationto peripheral lymphocytemetaphase chromosomes (Bachem, Philadelphia, PA); and GRO~ (a gift from M. P. Beck- of a 46, XY normal male with a biotin-labeled genomic 6.5-kb mann, S. Lyman, and D. Cerretti, Immunex Corp., Seattle, WA). XbaI fragment containing the MIP-lc~/RANTES receptor gene. The MIP-I~ and MIP-1B used in the initial ligand screen (see Fig. 2 A) was a gift of U. Siebenlist (National Institute of Allergy and Infectious Diseases) and was used as a diluted supernatant of Sf9 Results and Discussion insect cells expressingimmunoreactive recombinant human MIP-lc~ (from clone pAT464) or MIP-1B (from clone pAT744) (20). All Activation oS PMN by MIP-lol and RANTES. The o~ proteins were diluted from aqueous stock solutions or culture su- chemokines have previously been shown to mobilize calcium pernatants into ND96 oocyte media (96 mM NaC1, 1 mM KC1, in PMN (1-3). The ~ chemokines MIP-lc~ and RANTES 1 mM MgC12, 1.8 mM CaC12, pH 7.45) containing 0.1% BSA. were also able to elicit rapid, transient elevations of [Ca2+]i Radioligand Binding Assay. Carrier-flee recombinant human in human PMN (Fig. 1 A) with ECs0 of 5 and 50 aM, MIP-lc~ (10 /~g) was labeled using 5 mCi NanSI (Amersham respectively (Fig. 1, B and C). In contrast, MCP-1 elicited Corp.) in 100/~10.2 M sodium phosphate, pH 7.2, and 50 #1 recon- an elevation in [Ca2+]i in PBMC (Fig. 1 A, traces e and J) stituted Enzymobeads (Bio-Rad Laboratories, Richmond, CA) as but not in PMN (Fig. 1 A, trace d). These data suggest that previously described (5). The specific activity was 300 Ci/mmol. The labeled protein migrated as a single band on a 14% polyacryl- PMN possess receptors for MIP-lc~ and RANTES but not amide gel and was functionally active (not shown). Single oocytes MCP-I. Crossdesensitization experiments performed with were incubated with nSI-MIP-lcr for 30 min on ice in 10 #1 of MIP-lc~, RANTES, and MCP-1 with PMN and PBMC sug- binding buffer (HBSS with 25 mM Hepes, 1% BSA, pH 7.4). Un- gested that putative PMN receptors for MIP-lol and RANTES bound ligand was removedby centrifugation of the oocyte through may be identical (Fig. 1 A). 300 #1 of PS0 siliconefluid (General Electric, Waterford, NY). The Cloning of cDNA for the MIP-lcr/RANTES Receptor. tubes were quickly frozen and 3, emissions from the amputated During the cloning ofcDNA for human IL-8R B (5), a related tips were counted. but distinct class of four HL-60 neutrophil cDNAs was ChromosomalLocalization. For primary assignment, a panel of identified. The longest cDNA of this group was designated 11 Chinese hamster x human hybrid cell lines were used (21). For regional localization, four rodent x human hybrid cell lines were p4. The p4 cDNA is 2,165 bp in length; the longest open used that contained either the short arm or the long arm of chro- reading frame is 1,065 bp, encoding a protein of 355 amino mosome 3 in the absence of an intact chromosome 3. DNA from acids The proposed codon for initiation of translation and somatic cell hybrids and controls were analyzed by PCR using the its flanking sequence is GGGATGG, which conforms favor- open reading frame primers. The PCR conditions were 95~ ably to the consensus rules (23). The 5'- and 3'-untranslated 5 min; then 30 cycles of 94~ I min; 55~ 1 min; 72~ I min. regions are 102 and 998 bp long, respectively. A poly(A) tail Fine mapping was performed as described (22) by fluorescence in is present.

SO0. A B INeutrophilsl j

o MIP la MIP-la ~, II b __ :+__ cc ]/ .... -10 -9 .4 -7 4) Log [IdlP-la] (M) :D Figure 1. MllXlc~and RANTES elicit transient elevationof [Ca2 + ]i r in human leukocytes. (A) PMN (traces a-d) or PBMC (traces e-J) (D were loaded with Indo-1 and then 0 b. C stimulated at the indicated times u. ! i i i f t 110. with buffer, 10 aM MIP-lo~, 100 I= f aM RANTES, or 50 aM MCP-1. IMononu Ceflsl I= Alterationsin the bound/freeratio o m reflect changes in the [Ca2+]i as de-

0 ~ T i i ) F p scribed in Materials and Methods. (/3 and C) Ligandconcentration de- MIP-lo ~. MOP'-T | pendenceof calciummobilization in 80, PMN by MIP-I~ and RANTES. Data are representativeof four sep- I I ! ! ! ! ~ . , . , . , , 0 +00 200 300 400 soo .w 4 4 -7 4 arate experiments with MIP-I~, Log[RANTES](M) two with RANTES, and one with Time Ks) MCP-1.

1423 Gao et al. Figure 2. Functional expression of the MIP-lo~/ ,o, T B 9 MIP-lcr p o RANTES / RANTE$ z~ceptor in Xenopus oocytes. (A) The p4 cDNA [] MCP-1 encodes a receptor selective for a fl chemokine. 5 d after injection with 10 ng of either p4 cRNA or "6 0 (filled bars) ~2o. IL-SR B cRNA (open bars), oocytes were stimulated with human rlb-8, GROom, or NAP-2 at 500 nM, or a 1:5 dilu- E ILl 10. tion of an SD supernatant containing recombinant human MIP-I~ or MIP-lfl. Neither MIP-lcx nor MIP-lfl acti- ,~ o vated IL-SR B. (B) The p4 cDNA encodes a promiscuous 0 receptor for MIP-lcx and RANTES. Oocytes were injected 910 -9 -8 -7 -iS with 10 ng p4 cRNA and stimulated 3 d later with the '~ l~ Log plgandI (U) I indicated recombinant human B chemokines. Oocytes that I IL41FIB i , p4 had been injected with 50 nl of water did not respond to MIP-lcx, RANTES, or MCP-1 over the same concen- 3m, tration range (not shown). The SEM was <10% of the E C mean value. Data are from a single experiment represen- 8O tative of four separate experiments. In A and B, the data "~ 2000. o are derived from five to eight replicate determinations per v point. Basal amounts of calcium efl]ux and calcium up- U take were similar for all experimental conditions. (C and .~ ,o0o .o 4O ss D) Binding of 1~I-MIP-Ir to oocytes injected with p4 2O cRNA. (C) Total (filled circles) and nonspecific (open circles) binding was determined by incubating oocytes injected 0 .Q 1.# .... with p4 cKNA with the indicated concentration of radio- -12 -11 -10 -9 41 -7 . ~ -10 -8 -6 -4 ligand in the absence or presence of a 100-fold molar excess Log [mI-MIP-leq (M) log IMIP-I~| (M) of unlabeled MIP-lc~, respectively. Nonspecific binding was subtracted from total binding to determine specific binding (open squares). Specific binding of ~2SI-MIP-la to oocytes expressing IL-SR B was undetectable. (D) Oocytes injected with p4 cR.NA were incubated with 100,000 cpm 12SI-MIP-1ot in the presence or absence of the indicated concentration of unlabeled MIP-lc~. 100% represents a mean of 6,401 cpm. Data in C and D are derived from triplicate determinations per point.

To determine whether IL-8 or a related molecule could phocytes activated with PHA, the transformed line activate the p4 product, Xenopus oocytes were injected with Jurkat, EBV-transformed B cells (Fig. 3), and from 10 non- p4 cRNA and calcium efflux was measured in response to hematopoietic tissues (not shown). This broad expression pat- a test panel of ligands. Oocytes injected with p4 cRNA ac- tern in mature and immature cells of the immune system is quired responsiveness to MIP-la and RANTES but not to unique among all known chemoattractant receptors and, to MIP-1B, MCP-1, or any of three ot chemokines that were tested (Fig. 2, A and B). The ECs0 for P.ANTES was •50 nM, similar to that observed for activation of a calcium flux response in PMN by this chemokine (Figs. 1 C and 2 B). The oocyte response to MIP-lc~ is specific for the entire con- centration range tested. However, it has two phases, one that appears to saturate at 100 nM MIP-lo~ and a second that did not reach a plateau at 5,000 nM MIP-la (not shown). The first plateau coincides with that demonstrated for PMN over this concentration range (Figs. 1 B and 2 B). The threshold for detection of specific binding of 1251- MIP-lcx to oocytes injected with the p4 cRNA was the same as that required for stimulation of calcium efflux. 12sI- MIP-lo~ did not bind specifically to oocytes expressing human IL-8R B. Specific binding was also detected in COS-7 cells transfected with p4 in the vector pcDNAI, but not in un- transfected COS-7 cells (not shown). However, at high con- centrations the ligand formed aggregates causing a high back- ground of nonspecific binding that precluded determination of the binding constant. Figure 3. Distribution of mRNA encoding the MIP-I~/RANTES Tissue Distribution of the MIP-lcr/RANTES Receptor. A receptor. Each lane contains 10/Lg of total cellular RNA from the indi- p4 probe hybridized with a single 3-kb band on blots of RNA cated source with the exception of the lane designated HL60 poly(A) + , from the human myeloid precursor cell lines HL-60, U937, which contains 10/zg of polyadenylated RNA from undifferentiated Hb60 cells. The blot was hybridized with a p4 probe and was washed at 68~ and THP-1, from HL-60 cells differentiated with dibutyryl in 0.1x SSPE for 1 h. The blot was exposed to XAR-2 film in a Quanta cAMP (HL-60 neutrophils), and from human B lymphocytes. III cassette at 80~ for 6 d. The position of ribosomal RNA is indicated Transcripts were not detectable in total RNA from T lyre- with arrows.

1424 The Human Macrophage Inflammatory Protein lcffRANTES Receptor our knowledge, all other types of receptors, particularly by ceptors and US28 all have addic NH2-terminal domain; the virtue of the abundant expression in B lymphocytes. We have net charge for IL-SR A and 13, the MIP-lcffRANTES not tested monocyte RNA by blot hybridization but instead receptor, and US28 is -9, -11, -6, and -6, respec- have cloned MIP-lcffRANTES receptor cDNAs from an tively. This is very uncommon in other G protein-coupled endotoxin-stimulated human monocyte library (Ahuja, J. K., receptors (P. M. Murphy, unpublished analysis). It is note- and P. M. Murphy, unpublished data). worthy that MIP-lc~ and RANTES, like other chemokines, The distribution of MIP-lot/RANTES receptor transcripts are basic polypeptides and could bind via charge interactions differs from the pattern expected based on the functionally with the extraceUular NH2-terminal domain of the receptor. defined targets of RANTES action in two regards. First, In contrast to the IL-SKs the NH~-terminal sequences of RANTES is known to attract "memory" T lymphocytes (7), the MIP-lodRANTES receptor and US28 are surprisingly but total RNA from peripheral blood T cells activated with similar (56% identity), further highlighting the special evolu- PHA did not contain detectable transcripts. Thus: (a) either tionary relationship of these two products. Cytomegalovirus the receptor gene is expressed at very low constitutive levels is known to infect myeloid and lymphoid cells in vivo (26), in T cells; (b) the receptor gene is turned offby PHA treat- cell types that express the MIP-lodRANTES receptor. Others ment of the cells; or (c) the RANTES receptor of T cells have speculated that US28 was acquired by viral hijack of is distinct from the receptor we have described. Second, MIP- a human gene (25). Thus, the hijacked human gene may be lc~/RANTES receptor transcripts are expressed at high levels for a/~ . We speculate that expression of in B cells, a cell type that reportedly is not a target for US28 may alter the responsivenessof infected lymphoid and/or RANTES action (7). Perhaps the cellular environment in myeloid cells to MIP-loe, R.ANTES, or another ~ chemokine. which the receptor is expressed determines the precise effector In this way the intraceUular environment may be rendered function that can be activated. more favorable for viral replication and/or establishment of Structural Comparison with the IL-SR and with CMV-US28. the latent state. Viral hijack of immunoregulatory ligand and The MIP-lot/RANTES receptor contains seven hydrophobic receptor genes has been reported for pox and herpes viruses. segments predicted to span the plasma membrane, a signa- These and other viruses may use the viral homologues to ture of receptors that are known to be coupled to heterotri- elude the immune system by molecular mimicry (reviewed meric G proteins (24). As expected, its closest known struc- in reference 27). We have cloned US28 by PCR from fibro- tural homologues are human IL-SR A (31% amino acid blasts acutely infected with human cytomegalovirus. No cross- identity) and B (33% identity); the lengths of all three hybridizing human genes were detected by blot hybridiza- receptors are "~355 amino acids (Fig. 4). Interestingly, the tion of human genomic DNA with a full-length US28 DNA open reading frame US28 of human cytomegalovirus (25) probe under low stringency conditions (final wash at 45~ encodes a protein that is similar in sequence to the MIP-lc~/ in 5 x SSPE; data not shown). RANTES receptor (33% amino acid identity). Analysis of the MIP-loe/RANTES Receptor Gene. Anal- The NHz-terminal segment is poorly conserved for even ysis of human genomic DNA by blot hybridization with a closely related G protein-coupled receptors, including IL-8R p4 probe under high and low (45~ 5 x SSPE) stringency A and B (4-6). Not surprisingly the MIP-lot/RANTES conditions indicated that the MIP-lot/RANTES receptor must receptor has limited sequence identity with the IL-SRs in be encoded by a small, single-copygene that lacks dose homo- this region. Nevertheless, the three known chemokine re- logues in human (Fig. 5 A). Thus, unshared receptors selec-

I II 20 40 60 80 100 IL-8RB MS~pQMNDFDDL~;MPPIDED YSP CMLETET ~ I IA- YALVFLLSLLGNSLVMLVI L Y SRVGR~VTDVY LLNLALAD LLFALT LP I W~ W I FG~ L I : I I ::I II I : I III: I II II II: : I::IIIIII:IIII III I I I II MIP- I(IR ~T~EDYEr~Tx-~e-DYG ...... DATP CQKVNER&FGAQL LP PLY S LVFV I GLVGN I LVWLVLVQYIqRLKNMTS I YLLNLAI SDLLFLFTLPFW I DTKLKDDWVFGDAM I II:II IIIIII ::IIIII : II II: I II II : :I ::I III :IIII III I I I CMV-US28 M-TPTTTTA-ELTTEFDY ..... DEDATPCVFTD%'LNQSKPVTLFL YGVVFLFGS I GNFLVI FT I TWRRR I QCSGDVI'F I NLAAADLLFVCTLP LWMQYL~ ~ S - ~ III IV V 120 140 160 180 200 220 CKVVSL ~YSGI LLLAC I SVDRYLAIVHJLTRTLTQ~R- HLVKIeVCLGCWGLSM~TLS LP FFLFRQAYHP ~PV~EVL~~ LP HTF~ I ~ ~ ~T ~ II I : II I : IIIIIIIII I I I I I : I : I : II: II II II : I CKI L~YSEI~ I I LLTI DR~VFJ~T~GVITS I I IW~ Z ~ GL~'PS~qT~I~I~2SL~S~F~~I I ~ I IKI L~ I I I: II : III III I I I II I I I : : : I I :I II I II I :I CTLL TACF YVAMFASLCF I TE IALDRYYAI VYM .... RYI~FVKQACLFS I FWWIFAVI IAI PHF~NQ- .... (DfTDYDYLEVSYP I I LNVELMLGAFVIP LSVI SYCYYRI SRI~rAVSQ

240 VI 260 280 VII 300 320 340 M~HRAMRVIFAVVL IFLLCWLPYI4LVLLAD~TQVI QE SCERRNNI GR~LDATE ILGFLH SCLNP I IYAF I GQNFRHGFLKI LAM .... E GLVSI~.FLARHR~ SYTS S SVNVS SNL 350 I :1 I:11::::11 I I:1111:1 ::: : I:11 :1: II : : I I:11 IIII I II I : I1: I : I I I~J(]qSKAV~L IFVI MI IFFLFWTP YNLT I L I SVFQDFLFTHE - CEQSRRLDLAVQVTEV I AY TH CCVNPV I yAFVGERI~RKTLRQL FIIRRVAVHLVKWLP FL ~SS TSP S~L ~ I : I1: :::: I I1:11 I1: ::: : :11 I I :1: II :1:II1:11 I III III I : I : : SRHKG~% IVR~rL IAVVLVF i i FWLP YHLTLFVDTLKLLKW I SS ~S~ I L TE SLAF CH CCLNPL LYVFVG-~-~ ...... yTVCWP SFAS DSFPAMYP GTTA 323 Figure 4. Primarystructure of the human MIP-lot/R.ANTESreceptor (MIP-lotR) and alignment with that of human IDSIL B (5) and CMV-US28 (25). Verticalbars indicateidentical residues for each adjacentsequence position; colons indicateresidues that are identicalfor US28 and IL-8R B but differ from MIP-lc~R. The locations of predicted membrane-spanningsegments I-VII are noted. Open boxes designatepredicted sites for N-linked glycosylation.Arabic numbers abovethe sequenceblocks referto the MIP-I~.R sequenceand are leftjustified. Dashesindicate gaps that wereinserted to optimize the alignment. The DNA sequenceof p4 has been submitted to GenBank under accessionnumber L10918.

1425 Gao et al. Figure 5. Analysis of the human MIP-lce receptor gene. (A) A blot of human genomic DNA digested with the indicated restriction endonucleases was hybridized with the p4 cDNA (washed at 68~ in 0.1x SSPE), The blot was exposed to XAK-2 film in a Quanta III cassette at -80~ for 6 d. DNA markers are in kilobases at the left. No crosshybridizinggenes were de- tected under the low stringency condi- tions (washed at 45~ in 5x SSPE). The open reading frame of the MIP-lo~/ KANTES receptor gene has a restric- tion site for EcoRI and for Pstl. (B) Chromosomal localization. Ideogram of G-banding pattern of human chromo- some 3 with vertical bars illustrating the regions present in somatic cell hy- brid lines. PCK amplificationresults are summarized below: +, hybrid positive; and -, hybrid negative for human MIP-Io~/RANTES receptor sequence. Bracket labeled SCH shows localization of the gene based on hybrid cell map- ping data. Bracket labeled FISH indi- cates localization of fluorescent in situ hybridization signals at 3p21. tive for MIP-lc~ and/or RANTES, if they exist, must be struc- matopoiesis have not yet been reported. It will be important turally quite divergent from the receptor described here. The to study whether all or only some of the known effects of 6.5-kb XbaI fragment seen in Fig. 5 A was cloned and se- MIP-lol and KANTES are mediated by the receptor described quenced. An intron-exon boundary is found at nucleotide here. If so, the structure of this receptor suggests that the -12 relative to the ATG initiation codon, but the open reading signal transduction mechanism(s) for these disparate effects frame and 3' flanking sequenceslack introns, a common finding will involve G protein(s). for G protein-coupled receptor genes. By using primers specific The MIP-lcffRANTES receptor is the first receptor for for the human MIP-lodRANTES receptor cDNA for PCR a hematopoietic growth regulator in the superfamily of amplification of DNA from rodent x human somatic cell receptors with seven transmembrane domains. Identification hybrid mapping panels, we have localized the gene to the of the human MIP-lodRANTES receptor will now facili- short arm of chromosome 3. Independent confirmation of tate precise delineation of the signal transduction pathways this assignment and further sublocalization of this gene to by which MIP-lot and RANTES activate responsive cells of band 3p21 was accompanied by fluorescence in situ hybrid- the hematopoietic system and may lead to new insights re- ization (FISH) of the 6.5-kb genomic XbaI fragment con- garding the pathophysiology of fever and wound healing as taining the gene (Fig. 5 B). The IL-SR genes are clustered well as myelopoiesis and chemotaxis. Moreover, the relation- on human chromosome 2q34-q35 (19). ship of the MIP-lo~/RANTES receptor and US28 will permit MIP-lot has been shown to regulate the growth of stem the investigation of new hypotheses regarding the pathogen- cells and other more mature myeloid precursors (12-15). The esis of human cytomegalovirus infection. effects of related chemokines, including KANTES, on he-

This work was supported by the National Institutes of Health grant R01 HG-00298 (to U. Francke) and the Howard Hughes MedicalInstitute, of which U. Franckeis a investigator and X. Li is an associate. Address correspondenceto Philip M. Murphy, Laboratoryof Host Defenses, NIAID, Building 10, Room 11Nl13, National Institutes of Health, Bethesda, MD 20892. Receivedfor publication 25 January 1993.

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1427 Gao et al.