Identification of receptor binding and activation determinants in the N-terminal and N-loop regions of the CC chemokine eotaxin

Eotaxin is a CC chemokine that specifically activates the receptor CCR3 cau sing accumulation of eosinophils in allergic diseases and parasitic infecti ons. Twelve amino acid residues in the N-terminal (residues 1-8) and N-loop (residues 11-20) regions of eotaxin have been individually mutated to alan ine, and the ability of the mutants to bind and activate CCR3 has been dete rmined in cell-based assays, The alanine mutants at positions Thr(7), Asn(1 2), Leu(13), and Leu(20) show near wild type binding affinity and activity. The mutants T8A, N15A, and K17A have near wild type binding affinity for C CR3 but reduced receptor activation. A third class of mutants, S4A, V5A, R1 6A, and I18A, display significantly perturbed binding affinity for CCR3 whi le retaining the ability to activate or partially activate the receptor. Fi nally, the mutant Phe(11) has little detectable activity and 20-fold reduce d binding affinity relative to wild type eotaxin, the most dramatic effect observed in both assays but less dramatic than the effect of mutating the c orresponding residue in some other chemokines, Taken together, the results indicate that residues contributing to receptor binding affinity and those required for triggering receptor activation are distributed throughout the N-terminal and N-loop regions. This conclusion is in contrast to the separa tion of binding and activation functions between N-loop and N-terminal regi ons, respectively, that has been observed previously for some other chemoki nes.