Identification of a stretch of six divergent amino acids on the α5 helix of Gα16 as a major determinant of the promiscuity and efficiency of receptor coupling

Abstract

A broad repertory of G-protein-coupled receptors shows effective coupling with the haematopoietic G16 protein. In the present study, individual residues along the C-terminal α5 helix of Gα16 were examined for their contributions in defining receptor-coupling specificity. Residues that are relatively conserved within, but diverse between, the subfamilies of cloned Gα subunits were mutated into the corresponding Gαz residues. Six Girlinked receptors with different coupling efficiencies to Gα16 were examined for their ability to utilize the various Gα16 mutants to mediate agonist-induced inositol phosphate accumulation and Ca2+ mobilization. Co-operative enhancements of receptor coupling were observed with chimaeras harbouring multiple mutations at Glu350, Lys357 and Leu364 of Gα16. Mutation of Leu364 into isoleucine appeared to be more efficient in enhancing receptor recognition compared with mutations at the other two sites. Mutation of a stretch of six consecutive residues (362-367) lying towards the end of the α5 helix was found to broaden significantly the receptor-coupling profile of Gα16, and the effect was mediated partly through interactions with the β2-β3 loop. These results suggested that a stretch of six distinctive residues at the α5 helix of Gα16 is particularly important, whereas other discrete residues spreading along the α5 helix function co-operatively for determining the specificity of receptor recognition.