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 G16 protein. In the present study, individual residues along the C-terminal alpha5 helix of Galpha16 were examined for their contributions in defining receptor-coupling specificity. Residues that are relatively conserved within, but diverse between, the subfamilies of cloned Galpha subunits were mutated into the corresponding Galpha(z) residues. Six G(i)-linked receptors with different coupling efficiencies to Galpha16 were examined for their ability to utilize the various Galpha16 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 Galpha16. 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 alpha5 helix was found to broaden significantly the receptor-coupling profile of Galpha16, and the effect was mediated partly through interactions with the beta2-beta3 loop. These results suggested that a stretch of six distinctive residues at the alpha5 helix of Galpha16 is particularly important, whereas other discrete residues spreading along the alpha5 helix function co-operatively for determining the specificity of receptor recognition.
Department of Biochemistry, Molecular Neuroscience Center and Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, People's Republic of China.
Maurice K C
MK
Ho
Jasmine H P
JH
Chan
Cecilia S S
CS
Wong
Yung H
YH
Wong
D000595:Amino Acid Sequence / Q000235:genetics / Q000502:physiology; D000596:Amino Acids / Q000235:genetics / Q000502:physiology*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics / Q000502:physiology; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment
D000596:Amino Acids; D044385:GTP-Binding Protein alpha Subunits; D010455:Peptides; D043562:Receptors, G-Protein-Coupled