©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Promiscuous Chemokine Binding Profile of the Duffy Antigen/Receptor for Chemokines Is Primarily Localized to Sequences in the Amino-terminal Domain (*)

(Received for publication, April 25, 1995; and in revised form, August 14, 1995)

Lu Zhao-hai (1)(§) Wang Zi-xuan (1)(§) Richard Horuk (3) Joe Hesselgesser (3) Lou Yan-chun (1) Terrence J. Hadley (2) Stephen C. Peiper (1)(¶)

From the  (1)Departments of Pathology and (2)Medicine, Henry Vogt Cancer Research Institute, University of Louisville, Louisville, Kentucky 40292 and the (3)Department of Immunology, Berlex Biosciences, Richmond, California 94804-0099

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Duffy antigen (DARC) is a promiscuous chemokine receptor that also binds Plasmodium vivax. DARC belongs to a family of heptahelical chemokine receptors that includes specific (IL-8RA) and shared (IL-8RB) IL-8 receptors. Ligand binding specificity of IL-8 receptors was localized to the amino-terminal extracellular (E1) domain. To determine the basis for promiscuous chemokine binding by DARC, a chimeric receptor composed of the E1 domain of DARC and hydrophobic helices and loops from IL-8RB (DARC/IL-8RB) was constructed. Scatchard analysis of stable transfectants demonstrated that the DARC/IL-8RB chimeric receptor bound IL-8 and melanoma growth stimulating activity (MGSA) with Kvalues almost identical to the native receptors. The hybrid receptor also bound RANTES, MCP-1, and MGSA-E(6)A (which binds DARC, but not IL-8RB), but not MIP-1alpha, similarly to DARC. Ligand binding to DARC transfectants was unaltered by anti-Fy3, but inhibited by Fy6, which binds an epitope in the E1 domain. The epitope recognized by Fy3 was localized to the third extracellular loop by analysis of insect cells expressing chimeric receptors composed of complementary portions of DARC and IL-8RB. These findings implicate the E1 domain of DARC in multispecific chemokine binding.


INTRODUCTION

Interleukin-8 (IL-8)^1 and melanoma growth stimulating activity (MGSA) are members of a bipartite family of structurally related chemoattractant cytokines, known as chemokines(1) . The chemokines are characterized by the presence of four conserved cysteine residues and have been classified into two separate groups, dependent on whether the first two conserved cysteine residues are separated by an intervening amino acid (C-X-C) or whether they are adjacent (C-C)(1) . The C-X-C class members include IL-8, MGSA, and neutrophil-activating protein-2, while the C-C class includes RANTES, monocyte chemoattractant protein (MCP-1), and the macrophage inhibitory protein (MIP-1) proteins. C-X-C chemokines are strong chemoattractants and neutrophil agonists(2) , which up-regulate adhesion molecules (3, 4) that mediate granulocyte adherence to endothelial cells lining post-capillary venues (4) . By contrast, C-C chemokines, such as RANTES, MCP-1, and the MIP-1 proteins, are chemoattractants for leukocytes involved in more chronic inflammatory processes(1) .

Receptors for pro-inflammatory chemokines are members of a superfamily that have seven transmembrane-spanning helices and are coupled to guanine nucleotide-binding proteins (G-proteins)(5) . Complementary DNAs (cDNA) encoding a specific IL-8 receptor (IL-8RA) (6) and a shared receptor that binds both IL-8 and MGSA (IL-8RB) (7) have been molecularly cloned. These receptors do not bind to members of the C-C branch. Similarly, specific receptors for C-C chemokines have been cloned, but these do not bind members of the C-X-C branch(8, 9) . Recently, a promiscuous chemokine receptor (DARC), which binds C-X-C chemokines as well as C-C chemokines, was identified in erythrocytes(10, 11, 12) . This receptor, known as DARC, is identical to the Duffy blood group antigen(13) , which is a binding protein for the malarial parasite Plasmodium vivax(14) . However, since greater than 95% of Africans in endemic areas and approximately 70% of African-Americans lack expression of the Duffy blood group antigen(15) , this has raised some doubts about the possible physiologic significance of this receptor. These doubts have been somewhat allayed by the recent finding that DARC is ubiquitously expressed by endothelial cells lining post-capillary venules (16) in individuals who are positive or negative for the blood group antigen(46) . In essence, these findings provide a novel perspective into the potential relevance of DARC which is so clearly expressed in an anatomic site central to chemokine-induced leukocyte trafficking.

Although both the IL-8 receptors and DARC bind chemokines, their functions in normal and pathologic chemokine physiology are probably very different. For example, DARC lacks a DRY motif in the second intracellular loop(17) , which has been associated with G-protein coupling(18) , and stimulation of DARC transfectants with chemokine fails to reveal evidence of cytosolic mobilization of free calcium ions (19) , a characteristic feature of all other G-protein-linked chemokine receptors.

To delineate sequences responsible for the divergence between the ligand binding specificities of DARC and IL-8 receptors, chimeric receptors were analyzed for chemokine binding. The binding specificity of a hybrid receptor composed of the amino-terminal extracellular domain of DARC and the membrane-spanning helices and loops of IL-8RB resembled that of DARC. While a monoclonal antibody (mAb) to an epitope in the amino-terminal extracellular domain of DARC blocked chemokine binding, a mAb to an epitope in the extracellular loops had no effect. Thus, these data demonstrate that the amino-terminal domain of DARC contributes sequences critical for determining the repertoire of ligands bound.


EXPERIMENTAL PROCEDURES

Materials

I-IL-8, I-MGSA, I-RANTES, and I-MCP-1 (specific activity 2200 Ci/mmol) were obtained from DuPont NEN. Unlabeled IL-8, MGSA, and the MGSA mutant E(6)A were purified as described previously(11, 20, 21) , RANTES and MCP-1 were from Peprotech. Enriched human erythrocytes from outdated blood were obtained from Peninsula Blood Bank, Burlingame, CA. Reagents for electrophoresis were from Novex and FMC. All other reagent-grade chemicals were from Sigma. The Fy6 monoclonal antibody to the Duffy blood group antigen was kindly provided by Dr. Margaret Nichols as a hybridoma culture supernatant containing 22 µg/ml IgG(22) . Monoclonal antibodies to the IL-8RB were kindly provided by Dr. Jin Kim of Genentech.

Isolation of Erythrocytes

Human erythrocytes were isolated from whole blood using standard techniques(23) .

Cell Culture

K562 cells were obtained from the American Type Culture Collection and were maintained in RPMI 1640 medium containing 10% fetal calf serum and 50 µg/ml penicillin and streptomycin. Transfected K562 cells were maintained in RPMI 1640 medium containing 10% fetal calf serum and 0.2 g/liter hygromycin. For binding assays the cells were collected, washed three times with Hanks' balanced salts medium, and resuspended in the same buffer containing 1% bovine serum albumin, 20 mM Hepes, pH 7.4. Cell viability was assessed by trypan blue exclusion, and cell number was determined by counting the cells in a hemacytometer.

cDNA Synthesis and Sequencing

The cDNAs that encode DARC and the IL-8RB were obtained by reverse transcriptase PCR using total cellular RNA extracted from normal human kidney tissue and bone marrow, respectively. RNA templates were reverse transcribed from an oligodeoxythymidilate primer. The single-stranded cDNA was then used as a template for PCR. The primers used for DARC cDNA synthesis were Fyu (5`-TTCCCAGGAGACTCTTCCGG-3`) and Fyd (5`-ACTTTAATTCAGGTTGACAGGTGG-3`), and for IL-8RB synthesis were IL8RBu (5`-CATGGAGAGTGACAGCTTTGAAGA-3`) and IL8RBd (5`-AATGTGCTGTGAAGAGAAGGGAGG-3`). Amplification products of the appropriate size were cloned into the TA vector (Invitrogen, La Jolla, CA), and their nucleotide sequences were determined using standard dideoxy chain termination reaction.

Construction of Chimeric cDNA

The homology between the sequences encoding the last five amino acids of the amino-terminal extracellular domain of DARC and those encoding five amino acid residues in the amino-terminal extracellular domain of IL-8RB ending 20 residues prior to the first predicted transmembrane-spanning helix was exploited to design a pair of complementary oligonucleotides (Ch1 and Ch2), which could anneal to either the DARC (Ch1, antisense) or the IL-8RB (Ch2, sense) cDNAs (Ch1, 5`-AGTAGAAAAAAGGGCAGGGCAGAG-3`; Ch2, 5`-CTCTGCCCTGCCCTTTTTTCTACT-3`). Chimeric cDNA was generated in three steps using a DNA amplification approach(24) . A DNA fragment (cha) containing the sequence encoding the intact amino-terminal extracellular domain of DARC was made by PCR (primers: Fyu and Ch1) using a cloned DARC cDNA as template. Another DNA fragment (chb), encoding the last 20 amino acids of the amino-terminal extracellular domain, the transmembrane-spanning helices, and loops of IL-8RB, was made in PCR reactions that contained Ch2 and IL8RBd primers and IL-8RB cDNA as template. Finally, these two overlapping DNA fragments, cha and chb, served as templates to generate a chimeric hybrid cDNA encoding the DARC /IL-8RB using the Fyu and IL8RBd primer pair. DNA amplification was carried out with an initial ``hotstart'' interval of 5 min at 95 °C, 94 °C for 45 s, 45 °C for 45 s (ramp 5 min), and 72 °C for 1 min (ramp 5 min). The final amplification product was generated by 30 cycles composed of 45 s at 94 °C, 30 s at 60 °C, and 60 s at 72 °C.

Expression of DARC, IL-8RB, and Chimera DARC/IL-8RB

The DNA fragments encoding DARC, IL-8RB, and the DARC/IL-8RB chimera were subcloned in the HindIII/NotI sites of an episomal expression vector pREP4.HY. K562 cells were transfected with 2 µg of plasmid DNA using Lipofectin reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Stable transfectants were selected by their resistance to hygromycin (0.2 mg/ml). After 2 weeks of selection, transfected cells were stained with the appropriate monoclonal antibodies (DARC and DARC/IL-8RB: Fy6, IL-8RB: 10H2) and subjected to flow cytometric analysis. Briefly, transfected cells were harvested by centrifugation at 1300 rpm for 5 min and washed twice with PBS. Approximately 1 times 10^6 cells were resuspended in 50 µl PBS and incubated with antibody at room temperature for 45 min, followed by addition of fluorescein isothiocyanate-conjugated goat anti-mouse IgG, heavy and light chains (Southern Biotechnology Associates, Inc.), at a 100-fold dilution for another 45 min in the dark at room temperature. The cells were washed once with PBS following each antibody incubation. The cells were resuspended in 200 µl of cold PBS and analyzed by flow cytometry using an Elite flow cytometer (Coulter, Inc.). Parallel experiments were set up as controls by using non-transfected K562 cells and by incubating transfected cells with non-relevant mouse IgG. Transfectants expressing high levels of cell surface receptors were derived by fluorescence-activated cell sorting.

Wild type and chimeric chemokine receptors were also expressed in insect cells using a baculovirus expression system using strategies detailed above. DNA segments encoding DARC, IL-8RB, DARC/IL-8RB (DARC aa 1-66; IL-8RB aa 27-355), IL-8RB/DARC (IL-8RB aa 1-47; DARC aa 67-338), DARC/IL-8RB (DARC aa 1-131; IL8RB aa 117-355), and DARC/IL-8RB (DARC aa 1-209; IL-8RB aa 208-355) were cloned into the pVL 1393 baculovirus transfer vector utilizing BamHI and NotI sites in the polylinker. Constructs were co-transfected along with linearized AcMNPV DNA (Invitrogen, San Diego) into Sf21 cells and infectious recombinant baculovirus was isolated from culture supernatants. Infected Sf21 cells were stained with monoclonal antibodies to the appropriate chemokine receptor amino terminus, as described above, or with the Fy3 monoclonal antibody.

Receptor Binding Assays

K562 cells (1 times 10^6 cells/ml) stably expressing DARC, DARC/IL-8RB, or IL-8RB were incubated with I-labeled ligands (0.2 nM) and varying concentrations of unlabeled ligands at 4 °C for 1 h. The incubation was terminated by removing aliquots from the cell suspension and separating cells from buffer by centrifugation through a silicone/paraffin oil mixture as described previously(25) . Nonspecific binding was determined in the presence of 1 µM unlabeled ligand. The binding data were curve-fit with the computer program IGOR (Wavemetrics) to determine the affinity (K(D)), number of sites, and nonspecific binding.


RESULTS AND DISCUSSION

To identify the receptor domains responsible for the promiscuous chemokine binding of DARC, we made transfectants that express chimeric receptors composed of complementary portions of DARC and IL-8RB. Complementary DNA encoding DARC, IL-8RB, and a hybrid receptor containing the amino-terminal extracellular domain of DARC and transmembrane-spanning helices and loops of IL-8RB (DARC/IL-8RB) were prepared by DNA amplification. cDNA templates encoding the wild type receptors were reverse-transcribed from total RNA and amplified using primers corresponding to 5` sense and 3` antisense sequences following reverse transcription. These cDNAs, in turn, served as templates for the amplification of receptor domains for the construction of chimeras, as shown in Fig. 1. The amino-terminal extracellular domain of DARC was amplified using an upstream primer in the 5` non-coding region and a downstream primer (Ch1) corresponding to sequences complementary to those encoding seven amino acid residues located in the proximal portion of the amino-terminal extracellular domain at the interphase of the first transmembrane-spanning helix of DARC (residues 62-69) and IL-8RB (residues 27-34). Similarly, a segment encoding the transmembrane-spanning helices and loops of IL-8RB was amplified using an upstream primer (Ch2) containing sequences complementary to those present in the downstream DARC primer (Ch1) and a downstream primer in the 3`-untranslated regions. These two overlapping segments served as templates in a PCR reaction containing a 5` sense DARC primer and a 3` antisense IL-8RB primer, thereby generating a hybrid cDNA encoding a chimeric receptor. Clones encoding each receptor were verified by nucleotide sequence analysis, subcloned into an episomal vector, and introduced into K562 cells by lipofection. Following selection in hygromycin, transfectant pools were analyzed for receptor expression by flow cytometry using the appropriate monoclonal antibodies to detect DARC and DARC/IL-8RB (Fy6) and the IL-8RB (10H2). As shown in Fig. 2, transfectants with high level receptor expression were enriched by multiple rounds of fluorescence-activated cell sorting. Transfectants with high level expression were used for analysis of ligand binding.


Figure 1: Schematic representation of wild type IL-8RB and DARC and the chimeric receptor (DARC/IL-8RB) composed of the amino-terminal extracellular domain of DARC and the predicted loops and transmembrane-spanning helices of IL-8RB.




Figure 2: Flow cytometric analysis of wild type and chimeric chemokine receptor expression by K562 transfectants. Prior to chemokine binding experiments, K562 transfectants were stained with monoclonal antibodies to the amino terminus of the respective receptors (Fy6, DARC and DARC/IL-8RB; 2H10, IL-8RB) as described under ``Experimental Procedures'' ( . . . , specific antibody, -, myeloma protein control). All of the transfectants (IL-8RB, top panel; DARC, middle panel; and DARC/IL-8RB, bottom panel) showed high level expression of their respective receptors.



To identify the receptor domains in DARC that give rise to the promiscuous chemokine binding of the molecule, we compared the ligand binding profile and receptor binding affinity of the DARC/IL-8RB chimera with those of the parent receptors, DARC and IL-8RB. To facilitate direct comparison of binding affinities, the receptors were stably expressed in K562 cells, and receptor competition curves were generated over a wide concentration range of unlabeled chemokines (MGSA, IL-8, MGSA-E(6)A) (Fig. 3). Scatchard analysis of I-MGSA displacement binding demonstrated that DARC transfectants bound MGSA with a K(D) of 21 nM, which is consistent with that previously reported(26) . Equally, IL-8RB transfectants bound MGSA at high affinity, 9.8 nM, similar to that observed in neutrophils(27) . Scatchard analysis of I-MGSA binding to DARC/IL-8RB transfectants showed that the hybrid receptor bound ligand with an affinity of 18 nM akin to that observed for DARC. Receptor binding experiments with I-IL-8 were virtually identical to those obtained with MGSA (K(D) DARC 20 nM, IL-8RB 2.2 nM, and DARC/IL-8RB 5.5 nM).


Figure 3: Inhibition of binding of I-MGSA to K562 cells transfected with DARC (A), IL-8RB (B), and DARC/IL-8RB (C) by unlabeled MGSA, IL-8 and E(6)A. Cells (1 times 10^6 cells/ml) were incubated for 1 h at 4 °C with I-MGSA (0.2 nM) in the presence of increasing concentrations of unlabeled MGSA, IL-8, and E(6)A. The incubation was terminated by removing aliquots from the cell suspension and separating cells from buffer by centrifugation through a silicone/paraffin oil mixture as described previously(25) . Nonspecific binding was determined in the presence of 1 µM unlabeled ligand. The binding data were curve-fit with the computer program IGOR to determine receptor binding affinity (K).



The ligand binding profile of the DARC/IL-8RB chimeric receptor was further analyzed by receptor binding studies with an MGSA mutant, E(6)A, that has previously been demonstrated to bind DARC, but not IL-8RB(21) . Scatchard analysis of displacement of I-MGSA by MGSA-E(6)A revealed a K(D) of 28 nM for the hybrid receptor and 27 nM for DARC. In contrast, the K(D) for MGSA-E(6)A binding to IL-8RB was 476 nM, which was consistent with that previously reported (21) (Fig. 3). Although DARC and IL-8RB have virtually identical binding specificities for C-X-C chemokines, they are not highly related at the level of primary structure (17, 28) and DARC, in addition, binds chemokines of the C-C type(10, 11, 12) . The binding site on chemokines recognized by DARC is probably different from that recognized by IL-8RB, since mutation of the ELR motif does not abrogate binding to DARC(21) , as is observed for the IL-8RB(29, 30) .

The promiscuous binding repertoire of the DARC/IL-8RB chimeric receptor was further demonstrated by displacement of I-RANTES binding with C-X-C and C-C chemokines. IL-8 and RANTES were capable of displacing the binding of I-RANTES to K562 transfectants expressing the hybrid receptor and DARC (Fig. 4). As expected, cells transfected with the IL-8RB did not bind radiolabeled RANTES (data not shown). Since the C-C chemokine RANTES was able to specifically bind to the DARC/IL-8RB receptor chimera, we determined the affinity of RANTES binding to the hybrid by Scatchard analysis (Fig. 5). Scatchard analysis of I-RANTES binding to the hybrid receptor yielded a linear plot consistent with a single class of high affinity binding sites with a K(D) of 5.0 nM compared to a K(D) of 7.0 nM for binding to DARC (data now shown). Competition of I-MCP-1 binding with unlabeled MCP-1 resulted in augmentation of radioligand binding, an effect that has been previously reported with chemokines(31) .


Figure 4: Inhibition of I-RANTES binding to K562 cells transfected with DARC, and DARC/IL-8RB. Cells were incubated for 1 h at 40 °C with I-RANTES, as indicated above, in the absence of cross competing ligand (solid boxes) or with 100 nM concentrations of unlabeled IL-8 (open boxes) or RANTES (cross-hatched boxes).




Figure 5: Scatchard analysis of I-RANTES binding to K562 cells transfected with DARC/IL-8RB. Cells (1 times 10^6 cells/ml) were incubated with I-labeled RANTES (0.2 nM) in the presence and absence of increasing concentrations of unlabeled RANTES. The Scatchard plot shown here is representative of three separate determinations. Binding shown represents specific binding. Nonspecific binding was around 2% of total I-RANTES added and has been subtracted.



These findings implicate the amino-terminal extracellular domain of DARC as a critical region in determining the promiscuous ligand binding repertoire. Additional studies were performed to gain direct insight into the role of the extracellular loops in receptor binding. In the context of the Duffy blood group antigen, the monoclonal antibody Fy6 is directed against an epitope in the amino-terminal extracellular domain and is sensitive to proteolysis with chymotrypsin. A second antibody type, designated Fy3, recognizes a chymotrypsin-insensitive epitope expressed at the cell surface, which has not yet been located on the receptor.

Since the Fy3 epitope apparently resides on a domain of DARC other than the amino-terminal extracellular domain, the effect of the monoclonal antibody on the ability of the receptor to bind labeled chemokine ligand was determined. As shown in Fig. 6, the binding of I-MGSA to DARC-positive erythrocytes was inhibited by preincubation of target cells with Fy6 (50% inhibition at 100 pM). In contrast, preincubation of the erythrocytes with increasing concentrations of Fy3 (up to 175 µM final concentration) resulted in chemokine binding levels virtually identical to that observed in reactions containing equal amounts of a control monoclonal antibody to IL-8RB, which does not bind to DARC, as well as a non-immune mouse myeloma protein (data not shown).


Figure 6: Inhibition of MGSA binding to DARC by anti-Fy6 and anti-Fy3 monoclonal antibodies to the human Duffy blood group antigen. Erythrocytes (1 times 10^7 cells in 500 µl) were preincubated with increasing concentrations of anti-Fy6 or anti-Fy3 for 1 h at 4 °C. The cells were then incubated for another hour with 0.25 nMI-labeled MGSA in a final volume of 600 µl. The binding reactions were terminated as described previously. Nonspecific binding was determined by addition of 100 nM unlabeled IL-8 to the binding reactions. The effect of an antibody to IL-8RB on MGSA binding to erythrocytes was also determined.



The absence of inhibition of chemokine binding by the Fy3 monoclonal antibody, a sterically large molecule, suggests that the domain(s) that harbor the epitope may have an inconspicuous role in ligand binding. The region of DARC that harbors the Fy3 epitope was further investigated by testing Sf21 cells that express chimeric receptors for binding to Fy3. As shown in Fig. 7, flow cytometric analysis confirmed that Sf21 cells infected with a recombinant baculovirus encoding DARC reacted with Fy3, as well as Fy6 (panel A). Sf21 cells infected with the virus encoding DARC/IL-8RB lacked reactivity with Fy3 but expressed the chimeric receptor on the cell surface, as evidenced by binding of the DARC domain to Fy6 (panel B). Infection of Sf21 cells with the IL-8RB/DARC virus resulted in cell surface expression of a chimeric receptor that bound Fy3, localizing this epitope to the extracellular loops of DARC (panel C). The Fy3 epitope was further localized by parallel analysis of chimeric receptors containing the amino-terminal extracellular domain and first extracellular loop of DARC (DARC/IL-8RB) and the amino-terminal extracellular domain, first, and second extracellular loops of DARC (DARC/IL-8RB). Sf21 cells infected with recombinant baculoviruses encoding DARC/IL-8RB (panel D) and DARC/IL-8RB (panel E) expressed chimeric receptors on the plasma membrane that bound Fy6 but not Fy3. These data indicate that sequences in the predicted third extracellular loop are required for expression of the Fy3 epitope. Sf21 cells expressing IL-8RB also lacked binding to Fy3 (data not shown). The finding that a monoclonal antibody to the amino-terminal extracellular domain of DARC blocks ligand binding, whereas a monoclonal antibody to the third extracellular loop has no effect, further implicates the former in ligand binding and supports the finding of promiscuous chemokine binding to the chimeric receptor that contains the amino-terminal extracellular domain of DARC.



Figure 7: Analysis of insect cells expressing chimeric receptors for epitopes recognized by anti-Fy6 and anti-Fy3. Sf21 cells were infected with recombinant baculoviruses encoding DARC (panel A) or chimeric receptors composed of complementary segments of DARC and IL-8RB (panels B-E, see diagram). Cells stained with anti-Fy6, anti-Fy3, and a control mouse myeloma protein by indirect immunofluorescence on day 3 were analyzed by flow cytometry.



In contrast to other chemoattractant receptors, such as the N-formyl peptide receptor (32) and the C5a receptor(33, 34) which are also members of the serpentine receptor family, the amino-terminal extracellular domain of the IL-8 receptors have been shown to be major determinants in determining ligand binding specificity(35) . The repertoire and affinity of chemokine binding by DARC and the IL-8RB were analyzed to determine the domain(s) responsible for promiscuous chemokine binding. A chimeric receptor composed of the amino-terminal extracellular domain of DARC and the transmembrane-spanning helices and loops of IL-8RB were efficiently expressed on the cell surface and found to bind MGSA and IL-8, as well as RANTES, at high affinity. Moreover, this hybrid receptor bound MGSA-E(6)A, a mutant form of MGSA that lacks high affinity binding to IL-8RB but retains high affinity binding to DARC. Together, these findings indicate that sequences that confer a promiscuous ligand binding repertoire are localized to the amino-terminal extracellular domain of DARC.

The ligand binding pockets of the vast majority of seven transmembrane-spanning receptors, aside from receptors for glycoprotein hormones(36, 37) , and chemokines(5, 38) , involve hydrophobic segments predicted to be localized to intramembranous regions(32, 39) . Mutational analysis of receptors for C5a and N-formyl peptides, both potent chemoattractants for neutrophils, revealed that ligand binding domains involved sequences in the transmembrane-spanning helices(34, 40) . A two-site model of ligand binding, which includes transmembrane-spanning loops and the amino-terminal extracellular domain, has been proposed for the C5a receptor(33, 34) . These data are based on the finding that antagonists of C5a binding fail to inhibit the binding of peptides from the carboxyl terminus of C5a and that truncation of the amino-terminal extracellular domain of the receptor, and also protease digestion, abolish the binding of intact C5a, but not binding and receptor activation by carboxyl-terminal peptides(33, 34) .

Based on primary structure, DARC is as closely related to the endothelin receptors as it is to the IL-8RB. The three non-allelic forms of endothelins are potent vasoactive agents secreted by endothelial cells of veins and arteries that have 21 amino acid residues and two intrachain disulfide bonds(41) . Two receptors of different ligand binding specificity, designated type A and B, have been cloned(42, 43) . Analysis of the domains involved in endothelin binding reveals that the endothelin type A receptor, which is expressed primarily by smooth muscle cells, has a ligand binding pocket that includes sequences in transmembrane helices I, II, III, and VII. In contrast, the endothelin type B receptor, which binds all three endothelins at similar affinity and is expressed by endothelial cells, has a ligand binding pocket that involves sequences from transmembrane-spanning helices IV-VI(44) .

A similar receptor chimera approach has been used to define the domain(s) of the type B receptor for IL-8 that confer high affinity binding of MGSA(35) . Analysis of ligand binding characteristics of chimeras composed of reciprocal portions of IL-8RA and IL-8RB revealed that the amino-terminal extracellular domain was sufficient to confer high affinity binding to both IL-8 and MGSA to the IL-8RB/IL-8RA chimeric receptor. The reciprocal chimera, IL-8RA/IL-8RB, demonstrated high affinity binding limited to IL-8, indicating that the extracellular loops did not contribute to ligand binding specificity.

It is not clear whether the amino terminus of receptors for IL-8 contain all of the sequences necessary for high affinity chemokine binding. However, at least for the IL-8RA it has been demonstrated that residues in the amino-terminal extracellular domain and each of the extracellular loops, primarily cysteine residues, are required for high affinity ligand binding(45) . While several reports have used genetic approaches to study structure-function relationships in heptahelical receptors, this is the first demonstration of domains involved in chemokine binding using both genetic techniques and monoclonal antibodies to multiple topologic regions of the receptor.

The extracellular loops of chemokine receptors could function as a second contact point for chemokine binding that does not impart ligand specificity or as a polypeptide scaffold that interacts with the amino-terminal extracellular domain to generate a conformation of the latter permissive for high affinity binding. The finding that the binding of an Fy3 immunoglobin macromolecule to the third predicted extracellular loop of DARC failed to alter chemokine binding provides independent evidence to support the interpretation that the loops contribute to the overall conformation required to form a binding pocket, but not as a second contact point for binding. Thus, based on our findings and the above studies, we postulate that although the amino-terminal extracellular domain of DARC contains the primary structural motifs required for promiscuous chemokine binding, the extracellular loops of this receptor may also play a role through a cooperative interaction to form an extracellular structure with the appropriate conformation for binding to pro-inflammatory chemokine ligands. Since occupation of the binding pocket antagonizes the association of the receptor with the P. vivax polypeptide ligand(13) , these studies also may provide valuable insight into the pathophysiology of malarial invasion of erythroid cells.


FOOTNOTES

*
This work was supported by the Duggan Endowment for Oncologic Research and the Humana Endowment for Excellence. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors contributed equally to this work and should both be considered as first author.

To whom correspondence should be addressed: J. Graham Brown Cancer Center, 529 S. Jackson St., Louisville, KY 40292. Fax: 502-852-4946.

(^1)
The abbreviations used are: IL-8, interleukin-8; MGSA, melanoma growth stimulating activity; DARC, Duffy antigen/receptor for chemokines; MCP, monocyte chemoattractant protein; MIP, macrophage inhibitory protein; mAb, monoclonal antibody; PCR, polymerase chain reaction; PBS, phoshate-buffered saline; aa, amino acid(s).


ACKNOWLEDGEMENTS

We thank Dr. K. J. Kim for providing the monoclonal antibody to IL-8RB, Dr. Margaret Nichols and Dr. Dominique Blanchard for Fy6 monoclonal antibodies, Dr. Peter Byrne for Fy3 monoclonal antibodies, Monique Dougherty and Judy Hollkamp for assistance in the preparation of the manuscript, Abby Carden for assistance in preparation of figures, and Chris Worth for performing flow cytometric analyses.


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