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 causing accumulation of eosinophils in allergic diseases and parasitic infections. 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 alanine, and the ability of the mutants to bind and activate CCR3 has been determined in cell-based assays. The alanine mutants at positions Thr(7), Asn(12), 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 CCR3 but reduced receptor activation. A third class of mutants, S4A, V5A, R16A, and I18A, display significantly perturbed binding affinity for CCR3 while retaining the ability to activate or partially activate the receptor. Finally, the mutant Phe(11) has little detectable activity and 20-fold reduced binding affinity relative to wild type eotaxin, the most dramatic effect observed in both assays but less dramatic than the effect of mutating the corresponding 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 separation of binding and activation functions between N-loop and N-terminal regions, respectively, that has been observed previously for some other chemokines.
Mayer
M R
MR
Department of Chemistry, Indiana University, Bloomington 47405-0001, USA.
Stone
M J
MJ
D000409:Alanine / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D054413:Chemokine CCL11; D019742:Chemokines, CC; D016207:Cytokines / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
C515456:CCL11 protein, human; D054413:Chemokine CCL11; D019742:Chemokines, CC; D016207:Cytokines; D008024:Ligands; D013912:Threonine; D010649:Phenylalanine; D001216:Asparagine; D007930:Leucine; D000409:Alanine; D002118:Calcium