Characterization of binding between the chemokine eotaxin and peptides derived from the chemokine receptor CCR3

The CC chemokine eotaxin plays a predominant role in eosinophil trafficking in vivo by specifically activating the chemokine receptor CCR3. We have sc reened a series of synthetic peptides corresponding to extracellular region s 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 cons tant of 80 +/- 38 mu M. However, linear or cyclic peptides derived from the first and third extracellular loops of CCR3 did not bind to eotaxin. Linea r and cyclic peptides derived from the second extracellular loop precipitat ed upon addition of eotaxin, H-1-N-15 correlation NMR spectroscopy indicate d that an extended groove in the eotaxin surface, whose edges are defined b y the N-loop, 3(10)-helical turn, and beta(2)-beta(3) hairpin, is the most likely binding surface for CCR3-(1-35). NMR assignments for CCR3-(1-35) wer e obtained using two-dimensional and three-dimensional homonuclear NMR expe riments, N-15-Filtered TOCSY spectra indicated that the central region of C CR3-(1-35), surrounding the DDYY sequence, is involved in the interaction w ith eotaxin, This was supported by the observation that a truncated N-termi nal peptide (CCR3-(8-23)) binds to eotaxin with a dissociation constant of 136 +/- 23 mu M, only slightly weaker than the full-length N terminal pepti de. Taken together with previous studies, these results suggest that intera ctions between the N-loop/beta(3), regions of chemokines and the N-terminal regions of their receptors may be a conserved feature of chemokine-recepto r complexes across the CC, CXC, and C chemokine subfamilies. However, the l ow affinity of the interactions observed in these studies suggests the exis tence of additional binding regions in both the chemokines and the receptor s.