Identification of a stretch of six divergent amino acids on the alpha5 helix of Galpha16 as a major determinant of the promiscuity and efficiency of receptor coupling.

A broad repertory of G-protein-coupled receptors shows effective coupling with the haematopoietic G 1 6 protein. In the present study. individual residues along the C-terminal α5 helix of Gα 1 6 were examined for their contributions in defining receptor-coupling specificity. Residues that are relatively conserved within, but diverse between, the subfamilies of cloned Ga subunits were mutated into the corresponding Gα z residues, Six G t -linked receptors with different coupling efficiencies to Gα 1 6 were examined for their ability to utilize the various Gα 1 6 mutants to mediate agonist-induced inositol phosphate accumulation and Ca 2 + mobilization. Co-operative enhancements of receptor coupling were observed with chimaeras harbouring multiple mutations at Glu 3 5 0 , Lys 3 5 7 and Leu 3 6 4 of Gα 1 6 . Mutation of Leu 2 6 4 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 a5 helix was found to broaden significantly the receptor-coupling profile of Gα 1 6 , 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 Gα 1 6 is particularly important, whereas other discrete residues spreading along the a5 helix function co-operatively for determining the specificity of receptor recognition.