Characterization of Binding between the Chemokine Eotaxin and Peptides Derived from the Chemokine Receptor CCR3

Abstract

The CC chemokine eotaxin plays a predominant role in eosinophil trafficking in vivo by specifically activating the chemokine receptor CCR3. We have screened a series of synthetic peptides corresponding to extracellular regions of CCR3 for their ability to bind eotaxin. A peptide corresponding to the N terminus of CCR3 (CCR3-(1–35)) bound to eotaxin with a dissociation constant of 80 ± 38 μm. However, linear or cyclic peptides derived from the first and third extracellular loops of CCR3 did not bind to eotaxin. Linear and cyclic peptides derived from the second extracellular loop precipitated upon addition of eotaxin. 1H–15N correlation NMR spectroscopy indicated that an extended groove in the eotaxin surface, whose edges are defined by the N-loop, 310-helical turn, and β2–β3 hairpin, is the most likely binding surface for CCR3-(1–35). NMR assignments for CCR3-(1–35) were obtained using two-dimensional and three-dimensional homonuclear NMR experiments. 15N-Filtered TOCSY spectra indicated that the central region of CCR3-(1–35), surrounding the DDYY sequence, is involved in the interaction with eotaxin. This was supported by the observation that a truncated N-terminal peptide (CCR3-(8–23)) binds to eotaxin with a dissociation constant of 136 ± 23 μm, only slightly weaker than the full-length N-terminal peptide. Taken together with previous studies, these results suggest that interactions between the N-loop/β3 regions of chemokines and the N-terminal regions of their receptors may be a conserved feature of chemokine-receptor complexes across the CC, CXC, and C chemokine subfamilies. However, the low affinity of the interactions observed in these studies suggests the existence of additional binding regions in both the chemokines and the receptors. The CC chemokine eotaxin plays a predominant role in eosinophil trafficking in vivo by specifically activating the chemokine receptor CCR3. We have screened a series of synthetic peptides corresponding to extracellular regions of CCR3 for their ability to bind eotaxin. A peptide corresponding to the N terminus of CCR3 (CCR3-(1–35)) bound to eotaxin with a dissociation constant of 80 ± 38 μm. However, linear or cyclic peptides derived from the first and third extracellular loops of CCR3 did not bind to eotaxin. Linear and cyclic peptides derived from the second extracellular loop precipitated upon addition of eotaxin. 1H–15N correlation NMR spectroscopy indicated that an extended groove in the eotaxin surface, whose edges are defined by the N-loop, 310-helical turn, and β2–β3 hairpin, is the most likely binding surface for CCR3-(1–35). NMR assignments for CCR3-(1–35) were obtained using two-dimensional and three-dimensional homonuclear NMR experiments. 15N-Filtered TOCSY spectra indicated that the central region of CCR3-(1–35), surrounding the DDYY sequence, is involved in the interaction with eotaxin. This was supported by the observation that a truncated N-terminal peptide (CCR3-(8–23)) binds to eotaxin with a dissociation constant of 136 ± 23 μm, only slightly weaker than the full-length N-terminal peptide. Taken together with previous studies, these results suggest that interactions between the N-loop/β3 regions of chemokines and the N-terminal regions of their receptors may be a conserved feature of chemokine-receptor complexes across the CC, CXC, and C chemokine subfamilies. However, the low affinity of the interactions observed in these studies suggests the existence of additional binding regions in both the chemokines and the receptors. human immunodeficiency virus interleukin-8 monocyte chemoattractant protein CC chemokine receptor CXC chemokine receptor CX3C chemokine receptor reverse phase high performance liquid chromatography heteronuclear single-quantum coherence nuclear Overhauser effect spectroscopy rotating frame nuclear Overhauser effect spectroscopy total correlation spectroscopy regulated on activation normal T cell expressed A hallmark of inflammatory responses is the infiltration of the inflamed tissue by white blood cells. The migration of white blood cells from the vascular circulatory system to inflammatory loci is stimulated by a family of small secreted proteins called chemokines (1Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1984) Google Scholar,2Luster A.D. N. Engl. J. Med. 1998; 338: 436-445Crossref PubMed Scopus (3252) Google Scholar). Chemokines function by activation of chemokine receptors that are located in the leukocyte membrane and belong to the superfamily of seven transmembrane helix G-protein-coupled receptors (2Luster A.D. N. Engl. J. Med. 1998; 338: 436-445Crossref PubMed Scopus (3252) Google Scholar, 3Murphy P.M. Cytokine Growth Factor Rev. 1996; 7: 47-64Crossref PubMed Scopus (282) Google Scholar). In addition to their roles in mediating inflammatory responses, several chemokine receptors have been identified as cofactors for HIV-11 entry into CD4+ cells (4Berger E.A. Murphy P.M. Farber J.M. Annu. Rev. Immunol. 1999; 17: 657-700Crossref PubMed Scopus (1881) Google Scholar). Chemokine ligands for these receptors can inhibit HIV-1 infection by preventing the virus from binding to the receptors (4Berger E.A. Murphy P.M. Farber J.M. Annu. Rev. Immunol. 1999; 17: 657-700Crossref PubMed Scopus (1881) Google Scholar). Thus, an understanding of the interactions between chemokines and their receptors will stimulate the rational development of therapeutic strategies against inflammatory diseases and HIV. The more than 40 chemokines identified to date can be classified into four subfamilies based on their primary sequences. The CC, CXC, and CX3C chemokines all contain four absolutely conserved cysteine residues, the first two of which are separated by zero, one, and three amino acid residues in the respective subfamilies (5Bazan J.F. Bacon K.B. Hardiman G. Wang W. Soo K. Rossi D. Greaves D.R. Zlotnik A. Schall T.J. Nature. 1997; 385: 640-644Crossref PubMed Scopus (1697) Google Scholar, 6Zlotnik A. Yoshie O. Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3276) Google Scholar); the C chemokines have only the first and third of the conserved cysteines (7Kelner G.S. Kennedy J. Bacon K.B. Kleyensteuber S. Largaespada D.A. Jenkins N.A. Copeland N.G. Bazan J.F. Moore K.W. Schall T.J. Zlotnik A. Science. 1994; 266: 1395-1399Crossref PubMed Scopus (627) Google Scholar). Chemokine receptors are generally specific for chemokines of only one particular subfamily; hence, receptors are denoted by the prefixes “CCR,” “CXCR,” etc. However, most chemokine receptors can be activated by multiple (but not all) chemokines within the relevant subfamily (1Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1984) Google Scholar, 6Zlotnik A. Yoshie O. Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3276) Google Scholar). Similarly, many chemokines can activate more than one receptor. The molecular basis of chemokine-receptor recognition and specificity is currently not well understood. The three-dimensional structures of a number of chemokines have been determined by NMR and/or x-ray crystallography (reviewed in Ref. 8Stone M.J. Mayer K.L. Rothenberg M.E. Chemokines in Allergic Disease. Marcel Dekker, Inc., New York1999: 67-94Google Scholar). Although some interesting differences in quaternary structure have been noted, the structure of the monomeric unit (which is likely to be the functionally relevant species) is highly conserved and consists of an unstructured N terminus (before the first of the four conserved cysteines), an irregularly structured N-loop (following the second cysteine), a single turn of helix, a three-stranded antiparallel β-sheet (strands are designated β1, β2, and β3), and a C-terminal α-helix, which packs against one face of the β-sheet (8Stone M.J. Mayer K.L. Rothenberg M.E. Chemokines in Allergic Disease. Marcel Dekker, Inc., New York1999: 67-94Google Scholar). The N terminus and N-loop are tethered to the β-sheet by two conserved disulfide bonds. Site-directed mutagenesis has indicated that the N-loop region of chemokines is for receptor the N terminus is for receptor activation (1Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1984) Google Scholar, J. 266: Full Text PDF PubMed Google Scholar, K. A. M. J. 1997; PubMed Scopus Google Scholar, S. A. S. D. J. S. D. M. B. J. K. K. 1999; PubMed Scopus Google Scholar). The N-terminal regions of several chemokine receptors are involved in chemokine binding and specificity S. N. J. Full Text PDF PubMed Google Scholar, N. W. D. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Wang J. Murphy P.M. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). on these a has been for chemokine receptor activation K. A. M. J. 1997; PubMed Scopus Google Scholar, J. G. S. S. A. 1994; PubMed Scopus Google Scholar, M. J. 1996; PubMed Scopus Google Scholar, K. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In the first the N-loop region of the chemokine binds to the N-terminal region of the receptor. In the second the N terminus of the chemokine binds to a second on the located between or to the transmembrane a of the receptor and transmembrane In addition to the mutagenesis the first of is supported by of interactions of the CXC chemokine interleukin-8 and the CX3C chemokine with peptides corresponding to the N-terminal regions of their respective receptors 1994; 338: PubMed Scopus Google Scholar, Bazan J.F. 1999; PubMed Scopus Google Scholar, D. 1999; 7: Full Text Full Text PDF Scopus Google Scholar). In an NMR of interactions between the CC chemokine eotaxin E.A. Rothenberg M.E. J. A.D. Med. 1997; Scopus Google and peptides derived from the eotaxin receptor CCR3 M. S. Murphy P.M. Yoshie O. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). CCR3 is expressed on and and the of these cell upon activation of CCR3 by eotaxin is to a role in diseases and and in responses to D.A. O. T.J. J. Med. 1994; PubMed Scopus Google Scholar, M. J. 1996; PubMed Scopus Google Scholar, N. M. J. J. S. Dewald B. M. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, T.J. Med. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In to many eotaxin and M. M. M. J. Med. 1997; PubMed Scopus Google and M. N. S. K. Yoshie O. J. 1999; Full Text Full Text PDF PubMed Scopus Google are specific for their receptor CCR3 can be activated by the chemokines and which have receptor specificity A. Yoshie O. Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3276) Google Scholar, Wang J. W. J. Med. 1996; PubMed Scopus Google Scholar, G. N. J. 1997; PubMed Scopus Google Scholar). eotaxin has high to the CC chemokine which receptors and not CCR3 Immunol. PubMed Scopus Google Scholar, A. S. A. 1994; PubMed Scopus Google Scholar). Thus, studies of the interaction have the to chemokine-receptor In an to the eotaxin recognition of have screened peptides derived from of the extracellular regions of CCR3 N terminus and three for their ability to bind to cyclic of the extracellular loop peptides were a system for the and of the the NMR of within both eotaxin and the N-terminal receptor peptide involved in their and the of a peptide the primary binding of the receptor N A the amino acid of eotaxin E.A. Rothenberg M.E. J. A.D. Med. 1997; Scopus Google with the for in was by PubMed Scopus Google from The was and extended to the and for the and was into or in was expressed and as of involved of as by the cells with the were to of with and of eotaxin was The cell was in of and by The was and the was in of and The was and The was a affinity The was with and and with The protein was against The N-terminal was by with human of The protein was on a chromatography using a as the peptides were by the The peptides were by high performance liquid chromatography using a A of to was for peptide with a of loop peptides were first in for by the peptides were in and to was using as of and and Inc., and was within The cyclic loop peptides were by The molecular of the by were with the molecular NMR were on a were to and were to liquid J. J. PubMed Scopus Google Scholar). NMR were on a using Inc., for protein have been K. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Mayer K.L. M.J. J. 1999; 15: PubMed Scopus Google Scholar). peptide was to in loop loop and or CCR3-(1–35), loop and that the of the of peptide to eotaxin were and spectra were for 1994; 338: PubMed Scopus Google J. Scopus Google Scholar). for binding of CCR3-(1–35) and to eotaxin were from the of The observed of is by is the between bound and C is the total of and C is the of bound which is by C is the total of the peptide. A single and a for were by of the 1994; 338: PubMed Scopus Google as in The in the was determined by that the of the is the of the The peptide CCR3-(1–35) was in TOCSY and were three and with of by for and by for homonuclear and were with for and and for J. Scholar, J. 1998; Scholar, Nature. PubMed Scopus Google Scholar). The were for two-dimensional and for three-dimensional The was for in two-dimensional TOCSY and three-dimensional and with of for two-dimensional and for three-dimensional J. Scholar, J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The was by the in and and by both and a in the J. NMR and Scholar). were into the for coherence for the truncated peptide in the were using two-dimensional and spectra by eotaxin binding were identified by TOCSY spectra of CCR3-(1–35) in the of and of eotaxin. TOCSY was derived from a two-dimensional TOCSY by addition of of the and in which the of the was the to from of the The second was the and the J. NMR and Scholar); from to by The N-terminal regions of several have been in their J. 266: Full Text PDF PubMed Google Scholar, J. J. Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Med. PubMed Scopus Google Scholar). that the eotaxin in the was several of eotaxin with a N were the protein in A. of in which the N terminus the were low for and NMR studies than as by of cell In protein extended the N terminus by a expressed In a was the and the eotaxin to of the and and which are to be by the relevant the recognition to eotaxin with a N However, protein and of protein was of eotaxin by has been observed M.E. P.M. M. A.D. Med. 1996; PubMed Google Scholar). In protein which a recognition J. PubMed Scopus Google the N terminus of is and specifically by However, between the and of the recognition a acid the N A by K. J. 1998; Full Text Full Text PDF PubMed Scopus Google a acid N-terminal The results with indicated that the be to an N-terminal that be by to the eotaxin N In that the first amino acid of the eotaxin is the as the first amino acid the Thus, that of a recognition whose two amino are the first two amino of the eotaxin sequence, the in was high and and specific The the eotaxin sequence, as by N-terminal amino acid and and the in a In the NMR assignments to by K. J. 1998; Full Text Full Text PDF PubMed Scopus Google for synthetic eotaxin. We are not of the been for However, previous on the specificity of suggest that the may be of for proteins in which the N-terminal amino acid is J. PubMed Scopus Google Scholar). eotaxin by Chemokines are to bind to their receptors by with the extracellular regions of the receptors and with some of the transmembrane The extracellular regions the N terminus and three loops transmembrane helix and and and and extracellular of CCR3 that affinity for the eotaxin binding of four synthetic linear peptides corresponding to the N terminus and of the extracellular loops of CCR3 In the binding of three synthetic peptides corresponding to the CCR3 loop extended by and the N and C and by disulfide between the cysteine residues and was using two-dimensional correlation NMR spectra of eotaxin in the of of peptide. The N-terminal receptor CCR3-(1–35), of some residues in eotaxin observation is of on the of than the of eotaxin upon binding to CCR3-(1–35). The binding surface indicated by the of these residues is of the binding for binding indicated a dissociation constant of ± 38 linear peptides corresponding to loops and of CCR3 in the of eotaxin. Linear and cyclic loop peptides were in NMR was observed upon their addition to the eotaxin that is some interaction between loop peptide and eotaxin. In to the region of CCR3-(1–35) that with the NMR of the peptide and observed in peptide upon with eotaxin. The peptide was all the were in the the not be using the two-dimensional TOCSY and to three-dimensional homonuclear and together with the two-dimensional of the peptide. A a from the three-dimensional in which residues whose and are in the two-dimensional TOCSY can be by to in the three-dimensional a corresponding from the three-dimensional several to are well in The observed in two-dimensional and three-dimensional spectra of CCR3-(1–35) are in of the observed are with only two between and and between and The and the of or together suggest that CCR3-(1–35) is unstructured in However, the regions the two to to and to that these regions may some may their in peptide upon eotaxin were observed using The TOCSY was that the of be determined from their TOCSY to the in some regions of TOCSY of CCR3-(1–35) in the and of eotaxin are in The of most residues were upon binding to of several residues and by more than and as did the of one from were eotaxin In the bound peptide assignments with the protein the of that as for the structure of the D. 1999; 7: Full Text Full Text PDF Scopus Google Scholar). However, were by two the of the in many of the peptide or upon binding to eotaxin. The observed is more than be to the correlation of a to peptide. Thus, is that the peptide is by or on an that μm. The TOCSY that the central region of CCR3-(1–35) is for eotaxin a truncated peptide corresponding to residues to of CCR3 is in of the first conserved which is to a disulfide to extracellular loop in the receptor J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Full Text PDF PubMed Google Scholar). NMR of eotaxin with indicated that the truncated peptide binds to eotaxin with of 136 ± 38 only slightly than the for the full-length N-terminal peptide not In the in eotaxin observed upon binding to the truncated peptide have a to observed for the the of the are generally slightly suggest that the peptide binds to eotaxin in a to the peptide. The extracellular regions of G-protein-coupled receptors of ligands as peptides and proteins are in binding J. J. 1999; PubMed Scopus Google Scholar). In the of chemokine several of suggest that the N terminus of the receptor is an for chemokine receptors in which the N terminus of one receptor is by that of have been to specificity by the N-terminal N. W. D. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Wang J. Murphy P.M. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In receptors of the N terminus of to or receptor with single transmembrane bind to with to that of that the N-terminal of chemokine receptors may affinity for the ligands J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). have the of N-terminal peptides to the interactions between chemokines and their receptors. Thus, the N-terminal peptides of and have been by NMR to bind and 1994; 338: PubMed Scopus Google Scholar, Bazan J.F. 1999; PubMed Scopus Google Scholar, D. 1999; 7: Full Text Full Text PDF Scopus Google Scholar); the for the peptide interaction is 1994; 338: PubMed Scopus Google and the interaction is in the to Bazan J.F. 1999; PubMed Scopus Google Scholar). have K. and M. J. in that the N-terminal peptide of CCR3 binds to the CC chemokine with μm, and in the that the N-terminal peptide of CCR3 and a truncated of the N-terminal peptide both bind to eotaxin with affinity to observed for chemokine-receptor peptide complexes and μm, the peptide binding results that the N-terminal regions of CC, CXC, and CX3C chemokine receptors most likely roles in binding to their However, the low observed for these interactions that additional binding are for high affinity TOCSY for CCR3-(1–35) and the observed binding for that the central region of the CCR3 N-terminal is for affinity for eotaxin. is a particular of residues that upon eotaxin binding to the of the N-terminal peptide that these residues to the the that the of the receptor N-terminal region is conserved chemokine is between the N-terminal of chemokine receptors. many of the receptor N contain residues in to and/or residues in in in and in in the N-terminal of is and is for both chemokine binding and HIV-1 entry into cells M. M. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Similarly, of the residues in the of and the and of indicated that the residues are for chemokine binding S. A. S. D. J. S. D. M. B. J. K. K. 1999; PubMed Scopus Google Scholar, J. J. O. G. M. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The likely basis of the of these is indicated by the structure of the between and an N-terminal peptide from D. 1999; 7: Full Text Full Text PDF Scopus Google Scholar). The three residues of the with and residues, and the with a in D. 1999; 7: Full Text Full Text PDF Scopus Google Scholar). likely that the in chemokine receptors with the is that the of the residues within may be involved in specificity for particular A a of eotaxin to the observed upon binding to CCR3-(1–35). that the corresponding is to the binding or that the of that upon residues that are located in the N-loop and the 310-helical turn and and the and residues with than are and the and and the N terminus of the in and the N-loop, 310-helical turn, and the β2–β3 together an extended groove on one face of the eotaxin The of the groove is defined by residues and the edges of the groove are highly by and of the residues the groove were by the addition of the N-terminal with the of and for and were not from to the of their The surface of eotaxin is in the of the groove Thus, likely that the N-terminal receptor peptide in with the of the groove and interactions with residues on the edges of the This is in with the of the of CCR3 in chemokine The of observed for eotaxin is to observed for the CXC chemokine and the CX3C chemokine upon binding to N-terminal from their receptors 1994; 338: PubMed Scopus Google Scholar, Bazan J.F. 1999; PubMed Scopus Google Scholar, D. 1999; 7: Full Text Full Text PDF Scopus Google and to the of upon binding to CCR3-(1–35) K. and M. J. in Scholar). In the NMR structure of the between and the N-terminal peptide the peptide in an extended in the surface groove of D. 1999; 7: Full Text Full Text PDF Scopus Google Scholar). The groove in the CC chemokine has been as the binding for receptor S. A. S. D. J. S. D. M. B. J. K. K. 1999; PubMed Scopus Google Scholar). of several chemokines have indicated that residues in the N-loop region are for high affinity receptor binding J. 266: Full Text PDF PubMed Google Scholar, J. M. J. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, D.R. D.R. 1997; PubMed Scopus Google that the interactions observed in the peptide binding studies are Thus, likely that binding between surface groove of chemokines and the N-terminal region of their receptors is a conserved feature of chemokine-receptor the of interactions between the N-loop regions of chemokines and the N-terminal regions of their is low between the N-loop regions of Taken together with the low between receptor N suggests the that between these regions of the chemokine and receptor may an role in chemokine-receptor has a role for the of and J. M. J. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, D.R. D.R. 1997; PubMed Scopus Google Scholar). Although the N of chemokine receptors to be for chemokine is that peptides corresponding to the receptor N have low affinity for their The proteins in which the N terminus of is to or to the receptor suggest that the of the receptor N terminus to the cell membrane may be for high affinity binding J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). However, are of receptors in which the N terminus of a receptor was not to high affinity binding for chemokine Wang J. Murphy P.M. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Murphy P.M. J. 1996; Full Text Full Text PDF PubMed Scopus Google the that a second region of the chemokine receptor may be for high affinity This second region is most likely to be by one or more of the extracellular loops of the receptor may one or more of the transmembrane the chemokine into the transmembrane of the receptor. In an to regions of the CCR3 extracellular loops with affinity for screened both linear and cyclic peptides corresponding to of the extracellular from loops and effect on both linear and cyclic peptides corresponding to loop precipitated upon with eotaxin. The observed may be a are the N of the loop peptides and eotaxin is highly with an of The feature that loop is in amino is not chemokine receptors. However, the second extracellular loop of has been to a role in binding J. J. O. G. M. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the of the loop interaction Although the peptides derived from the CCR3 extracellular loops not specific binding to eotaxin in that the loops with eotaxin in the receptor. are two that affinity for the loop the peptides are be a for in the to bind eotaxin. binding of receptor regions to the chemokine is to be The conserved disulfide between the receptor N terminus and loop and between loops and J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1994; Full Text PDF PubMed Google may both to the loop and a for chemokine The of the loop peptide by their disulfide did not chemokine The results suggest that the extracellular loop regions are not involved in chemokine recognition or that observation of their interactions with chemokines will of the receptor or development of a system that multiple receptor extracellular with the for chemokine The that eotaxin binds to peptides derived from the N-terminal region of CCR3 with The binding surface of eotaxin to of an extended groove whose are defined by residues from the N-loop, 310-helical turn, and β2–β3 of the groove and peptide upon eotaxin binding are with a in which the and surrounding residues of the N-terminal peptide with residues the of the groove and/or the edges of the In with previous studies, these results suggest that a binding may to all chemokine-receptor Although several of suggest the of a second receptor region for high affinity chemokine with peptides from the CCR3 extracellular loops to a that interactions are and/or that transmembrane to the additional binding The binding by the results will be in and molecular