Cloning of a Human Seven-transmembrane Domain Receptor, LESTR, That Is Highly Expressed in Leukocytes*

Several chemotactic agonists including interleukin-8 (IL-8) and related cytokines have been shown to activate and attract leukocytes via seven-transmembrane domain, GTP-binding protein-coupled receptors. A cDNA clone, LESTR, encoding a protein of 352 amino acids, corresponding to a novel receptor of this type, was isolated from a human blood monocyte cDNA library. The sequence of the deduced protein, LESTR (leukocyte-derived seven-transmembrane domain receptor), has 92.6% identity with that of a recently reported bovine neuropeptide Y (NPY) receptor, boLCR1 (Rimland, J., Xin, W., Sweetnam, P., Saijoh, K., Nestler, E. J., and Duman, R. S. (1991) Mol. Pharmacol. 40, 869-875). LESTR, however, is more similar (> 34%) to the IL-8 receptors, IL-8R1 and IL-8R2, than to several NPY receptors of different origin (< 20%). In the monocyte library, LESTR cDNA fragments were about 20 times as frequent as cDNA coding for IL-8R1 and IL-8R2, and much higher levels of LESTR- than IL-8R-specific mRNA were found in human blood neutrophils and lymphocytes. LESTR transcripts, by contrast, were low or undetectable in several neuroblastoma cell lines that are widely used to study NPY functions. Transfected cells expressing high levels of LESTR mRNA did not bind radiolabeled NPY, IL-8, NAP-2, GRO alpha, PF4, IP10, MCP-1, MCP-3, MIP-1 alpha, HC14, I309, RANTES, C3a, or LTB4. NPY also failed to bind to neutrophils, monocytes, and lymphocytes, to elicit responses in vitro such as Ca2+ changes, shape change, chemotaxis, enzyme release, and the respiratory burst, and to induce leukocyte accumulation upon injection in rats and rabbits. Although the ligand for LESTR could not be identified among a large number of chemotactic cytokines, the high expression in white blood cells and the marked sequence relation to IL-8R1 and IL-8R2 suggest that LESTR may function in the activation of inflammatory cells.

IL-8R1 and IL-Sh, than to several NPY receptors of different origin (<200/0). In the monocyte library, LESTR cDNA fragments were about 20 times as frequent as cDNA coding for IL-8R1 and IL-Sh, and much higher levels of LESTR-than IL-8R-specific mRNA were found in human blood neutrophils and lymphocytes. LESTR transcripts, by contrast, were low or undetectable in several neuroblastoma cell lines that are widely used to study NPY functions. Transfected cells expressing high levels of LESTR mRNA did not bind radiolabeled NPY, HC14, 1309, RANTES, C3a, or LTB4. NPY also failed to bind to neutrophils, monocytes, and lymphocytes, to elicit responses in vitro such as Ca2+ changes, shape change, chemotaxis, enzyme release, and the respiratory burst, and to induce leukocyte accumulation upon injection in rats and rabbits. Although the ligand for LESTR could not be identified among a large number of chemotactic cytokines, the high expression in white blood cells and the marked sequence relation to IL-8R1 and IL-8b suggest that LESTR may function in the activation of inflammatory cells.
IL-8, NAP-2, GROa, PF4, IP10, MCP-1, MCP-3, MIP-la, The recruitment of leukocytes in inflamed tissues became a major area of research after the discovery of IL-8l and several * This work was supported by Grant 31-25700.88 from the Swiss National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankTMIEMBL Data Bank with accession number(s1
The nucleotide seguence(s) reported in this paper has been submitted M99293. $ To whom correspondence should be addressed. Fax: 41 31 631 3799.
Chemotactic agonists act via seven-transmembrane domain, GTP-binding protein (G-protein)-coupled receptors, as shown originally for met-Leu-Phe ( 5 ) and C5a (6). Two IL-8 receptors, IL-8R1 and IL-8R2, were characterized subsequently by biochemical, molecular biological, and functional studies (7-9). They bind all known chemotactic cytokines of the CXC subfamily, which are believed also to share the signal transduction system (1). High levels of transcripts for these receptors are found in neutrophils, while expression in other leukocytes is low (10). Chemotactic cytokines of the CC subfamily do not bind to IL-8 receptors. High affinity binding sites for monocyte chemotactic protein 1 (MCP-1) and RANTES have been found on monocytes (11-13).
In view of the similarities among the CXC and CC chemotactic cytokines, we assumed that their receptors are structurally more related to each other than to other G-protein-coupled transmembrane proteins, and we used an IL-8 receptor cDNA probe to isolate putative CC cytokine receptor cDNAs from monocytes. In this paper we describe the isolation of a novel cDNA encoding a seven-transmembrane domain protein, LESTR (leukocyte-derived seven-transmembrane domain receptor), that is closely related to the IL-8 receptors and has more than 90% identity with a bovine brain receptor for neuropeptide Y (NPY), boLCRl (14). Interestingly, this novel human monocyte protein is highly expressed in leukocytes, suggesting that it may be involved in the regulation of their function.
cDNA Cloning-Total RNA from human monocytes (>95% monocytes, 3 4 % lymphocytes) was extracted by the guanidinium thiocyanate5iCl method (18), and 5 pg of purified poly(A)+ RNA were used for cDNA synthesis (Riboclone cDNA synthesis system, Promega Hybridization was performed in 6 x SSC, 0.05 x Blotto (20) with lo6 c p d m l hybridizing solution at 68 "C for 16 h. The filters were washed once in 2 x SSC, 0.1% SDS at room temperature for 10 min. twice in 1 x SSC, 0.1% SDS a t 68 "C for 30 min, and twice in 0.5 x SSC, 0.1% SDS at the same temperature for 15 min. The cDNAs from 60 positive clones were further characterized by restriction enzyme site mapping, Southern hybridization, and partial sequence analysis. Two selected cDNAs of 1.2 and 1.6 kb were subcloned into the Gene Scribe-2 vectors, pTZ18/ 19/U/R (U. S. Biochemical Corp.) and sequenced to completion (22). As the nucleotide sequences of both clones were identical within the overlapping region, only the larger cDNA clone, LESTR, was used for further studies.
Northern Analysis-RNA samples of 10 pg were fractionated on denaturing formaldehyde-agarose gels (20), vacuum-transferred (trans-VAC TE 80, Hoefer Scientific Instruments) onto Nytran membranes (Schleicher and Schuell), and immobilized by baking at 80 "C for 2 h. A 500-bp internal XhoII fragment of LESTR, encoding the second half of the protein, was labeled with [CY-~~PICTP by random primed synthesis and used for hybridization at 2 x IO6 c p d m l i n t h e presence of 50% formamide a t 42 "C for 24 h (20). The membranes were washed once in 2 x SSC at room temperature for 20 min, two times in 0.25 x SSC, 0.1% SDS at 55 "C for 20 min, and once in 0.1 x SSC, 0.1% SDS at 55 "C for 20 min and exposed to Hyperfilm-MP (Amersham International plc) a t -70 "C for 1-2 days.
Subcloning and Mammalian Cell fiansfection-The complete monocyte cDNA insert of LESTR was cloned into the Sac1 site of the pSVL expression vector (Pharmacia). For the generation of CHO transfectants, 4 x lo6 cells were cotransfected by electroporation with 20 pg of pSVL-LESTR and 10 pg of pSV2,,,, both linearized with EcoRI. Neomycin-resistant CHO clones were selected by addition of G-418 (Life Technologies, Inc.) a t 500 pg/ml culture medium and screened for expression of LESTR by Northern blot analysis. Transient transfections in COS cells with pcDNAI-LESTR were performed by the DEAE-dextran method (5) or by electroporation (10).

RESULTS
Zsolation of LESTR cDNAs-The monocyte cDNA library was screened with a polymerase chain reaction probe encoding part of the rabbit IL-8 receptor (10,211. Out of 60 positive clones, 22 exhibited high hybridization intensities, as shown by Southern blotting. Only one clone for each of the two IL-8 receptors (IL-8R1 and IL-8R2) was identified, while all remaining 20 clones shared several common restriction enzyme digestion patterns. Partial sequencing demonstrated that their cDNA was derived from a single RNA species. Two clones of 1201 and 1645 bp were sequenced to completion. Both comprise the whole coding region and have identical nucleotide sequences. The clone with the larger insert (1645 bp), LESTR, was used for detailed characterization (Fig. 1). The longest open reading frame starts with a sequence that conforms well with the consensus sequence for initiation of translation in vertebrates (24) and encodes a polypeptide, LESTR, consisting of 352 amino acids with a calculated M, of

WSHTDKYRLBtSVADLLFVLTLPFWAV
LESTR shares 92.6% amino acid identity with boLCR1, a receptor encoded by a bovine brain cDNA (141, suggesting that LESTR is the human homologue of boLCR1. Moreover, LESTR has considerable homology to receptors for chemoattractants, the overall sequence identity between LESTR and IL-8 receptors (7, 8, 25) being about 34%. As shown in Fig. 2, extensive sequence identities are found in the region extending from the end of TM1 to the beginning of TM4, and within TM7. On the other hand, boLCRl and LESTR have only 15-20% amino acid identity with other cloned NPY receptors, i.e. human NPYRl from fetal brain, huNPYRl (311, rat NPYR from brain tissue, raNPYR (321, and Drosophila melanogaster NPYR, drNPYR (33).
Expression of LESTR-Several neuroblastoma cell lines as well as normal and transformed blood and tissue cells were analyzed by Northern blotting for expression of LESTR mRNA. An internal 500-bp XhoII fragment of LESTR encoding the second half of the receptor was used as hybridization probe. A predominant RNA species of 1.7-1.8 kb became labeled, indicating that LESTR represents a nearly full-length cDNA clone (Fig. 3). High levels of transcripts were obtained in human blood neutrophils and PBL, PHA-activated T cell blasts, and HL-60 cells differentiated with l~t,25(0H)~-vitamin Ds. In these cells LESTR expression was much more pronounced than expression of IL-8Rl/R2 (10). Moderate levels of LESTR tran- scripts were detected in blood monocytes and undifferentiated HL-60 cells, and only low levels in U937, Jurkat, and dimethyl sulfoxide-differentiated HL-60 cells. Among four different neuroblastoma cell lines that were analyzed, only two were positive: LAN5, a cell line derived from a bone marrow neuroblastoma metastasis, expressed high levels of transcripts, while SH-SY-BY gave only a faint hybridization signal. The abundance of LESTR mRNA in leukocytes is in agreement with the high incidence of LESTR-positive clones in our cDNA library derived from human blood monocytes.
Binding of Neuropeptide Y-Binding studies using radiolabeled NPY were performed with COS and CHO cells transfected with LESTR cDNA. The same cells were previously used to express boLCRl and showed high affinity binding ( nM) for 1251-NPY and low affinity binding for 1251-peptide YY, suggesting that boLCRl may correspond to the Y3 subtype of NPY receptors (14). The cDNA for the human NPY Yl-subtype receptor, huNPYR1, which was recently cloned, conferred high affinity binding (ICd 0.86 nM) for 1251-NPY when expressed in COS cells (31). CHO cells that stably express LESTR, as shown by Northern analysis, were generated by transfection of the corresponding cDNA (Fig. 4A). Binding assays were then performed using cells adherent to plastic or in suspension, as well as membrane preparations.
No specific binding to CHO cell transfectants was observed using 1251-NPY from two different manufacturers in experiments performed according to protocols where incubation time, temperature, and washing procedures were varied systematically. Fig. 4B shows the lack of specific high affinity NPY binding to CHO cell transfectants. Using the same 1251-NPY preparation, 26,000 siteskell with a Kd of 3 nM were identified on SK-N-MC neuroblastoma cells which bear NPY receptors of the Y1 subtype (34,35), demonstrating selective receptor recognition (Fig. 4B). Negative results were also obtained using COS cells that transiently expressed LESTR (not shown).
Dash, no activity detected in bioassays; no dash, assays not performed.
100 nM NPY was either added to the cells in the upper chamber or together with 0. 1-30 nM IL8 (neutrophils) or MCP-I (monocytes) into the lower chamber, and cell migration was assessed after 25-60 min a t 37 "C (41).

Since NPY did not bind to cells transfected with LESTR,
additional binding studies were performed with human blood phagocytes and lymphocytes that constitutively express high levels of LESTR mRNA (Fig. 3). As in transfected cells, no specific high affinity binding of NPY could be detected on neutrophils, monocytes, or lymphocytes (Table I). The limit of detection, in these experiments, was estimated to be 500 receptorskell (23).
Functional Assays with Neuropeptide Y-Because of the high level of expression of LESTR in neutrophils (Fig. 3), NPY was tested as potential agonist (Table I). At concentrations between and M, NPY was totally inactive in neutrophils that responded normally to IL-8 in the following functional assays: changes in cytosolic free calcium ([Ca2+li), shape change, chemotaxis, granule exocytosis (following pretreatment with cytochalasin B), and the respiratory burst (1). NPY was also inactive in human blood monocytes and lymphocytes, as indicated by the lack of [Ca2+Ij changes and chemotaxis in vitro. In addition, exposure of neutrophils and monocytes to NPY (lo-'-M for 1,20, and 40 min) did not affect the time course and extent of the [Ca2+Ii changes induced by subsequent stimulation with M IL-8 or M monocyte chemotactic peptide 1 (MCP-1) and met-Leu-Phe, respectively. Moreover, simultaneous incubation with M NPY had no measurable inhibitory influence on chemotactic activity in neutrophils and monocytes induced with optimal concentrations of IL-8 or MCP-1. Since IL-8 induces neutrophil infiltration when injected into animals (l), NPY was also tested in vivo. It did not induce the infiltration of neutrophils or other inflammatory cells in the skin of rats and rabbits and did not lead to accumulation of leukocytes into the cerebrospinal fluid as tested in a rabbit meningitis model (36).

Novel Leukocyte Seven-transmembrane Domain Receptor
MIP-la, RANTES, HC14,1309, as well as C3a, were iodinated and tested in binding studies. All ligands used in the binding experiments are listed in Table 11. None of them bound specifically to CHO cells expressing LESTR. DISCUSSION Clones containing fragments of a cDNA encoding a novel putative seven-transmembrane domain receptor, LESTR, were identified in a human monocyte cDNA library by screening with a probe specific for IL-8 receptors. LESTR is highly related to boLCR1, a recently described NPY receptor from bovine brain (14). Human myeloid cells expressed considerably higher levels of LESTR mRNA than transcripts for the two known IL-8 receptors, IL-8R1 and IL-8Rz (7, 8, lo), as shown by Northern analysis and by the incidence of LESTR clones, which was about 20-fold higher than for IL-8R1 and IL-8Rz clones in our cDNA library. By contrast, neuroblastoma cell lines that are used to study NPY functions (34,38) were negative or only weakly positive, except for LqN5 where LESTR expression was high.
The structural relationship to chemoattractant receptors and the high degree of expression in leukocytes suggest that LESTR may be involved in the regulation of leukocyte function. In the present study, however, we found no evidence for binding of NPY on transfected cells as well as human blood monocytes, neutrophils, or lymphocytes, which expressed high levels of LESTR mRNA, and no functional responses were induced in these cells by stimulation with NPY. In addition, NPY also failed to induce leukocyte accumulation and inflammation upon injection in rats and rabbits, indicating that this peptide does not activate inflammatory cells.
NPY exerts a variety of biological effects and has been implicated in the pathophysiology of diseases such as hypertension, congestive heart failure, and behavioral disorders (38). In brain and peripheral nerve tissue, three subtypes of NPY receptors, designated as Y1, Yz, and Y3, are distinguished on the basis of binding and function studies with NPY, peptide YY, and structural analogs (34). Ligand binding to all three receptor The CHO transfectants were produced and selected as described under "Experimental Procedures." All assays were performed with two CHO transfectant cell lines, 2D1 and 1C2, as described in Fig. 4B.
Positive controls were performed with cells known to bind the respective ligands. ND, not determined.
Dilution of lZ5I-RANTES with excess (1 p~) unlabeled ligands rebinding in the absence of unlabeled RANTES. Similar findings were sulted in a >'&fold increase in cell-bound radioactivity as compared with obtained by others (12, 45).
types leads to inhibition of adenylyl cyclase. This effect is prevented by pretreatment of the cells with Bordetella pertussis toxin, indicating that signal transduction is mediated by Gproteins of the Gi type (34,39,40). Several NPY receptor cD-NAs were cloned: huNPYRl from fetal brain (31) and raNPYR from brain tissue (32) belong to the Y1, drNPYR (33) to the Yz, and boLCRl to the Y3 type (14). Homology search in the Swis-sProt data base has shown that the LESTR sequence is only 15-20% identical with huNPYR1, raNPYR, and drNPYR. LESTR and consequently also boLCRl are more closely related to chemoattractant receptors, including those for Met-Leu-Phe, C5a, and platelet-activating factor, and share an overall sequence identity of 34% with IL-8 receptors (7,8,30). Despite this sequence similarity, CHO cells transfected with LESTR did not bind the chemoattractants IL-8, GROa, NAF"2, PF4, IPlO, MCP-1, MCP-3, MIP-la, RANTES, HC14,1309, C3a, and Recent Southern experiments with human genomic DNA under high stringency conditions using an internal 33011 fragment of LESTR revealed a hybridization pattern that corresponded to the presence of a single LESTR gene. Additional signals could not be detected, suggesting that the LESTR gene has no other homologues in the human genome and thus appears to be the most closely related homologue to boLCR1. It has been reported that boLCR1, like huNPYR1, binds NPY with high affinity although the sequence identity between these two receptors is only 18%. In view of these data, it was surprising to realize that LESTR, which is almost identical to boLCRl(92.6% true identity and 96.9% identity when conservative amino acid substitutions are considered), did not bind NPY. Few amino acid substitutions may suffice to alter the ligand specificity. Of a total of 11 non-conservative amino acid substitutions in LESTR with respect to boLCR1, seven are in the extracellular loops, one in TM4 and one in the N-terminal proximity of TM1. Site-directed mutagenesis of LESTR and boLCRl may be used to establish the structural requirements for NPY binding. LTBI.