Rhodopsin recognition by mutant G(s)α containing C-terminal residues of transducin

Abstract

The C-terminal regions of the heterotrimeric G protein α-subunits play key roles in selective activation of G proteins by their cognate receptors. In this study, mutant G(s)α proteins with substitutions by C-terminal residues of transducin (G(t)α) were analyzed for their interaction with light-activated rhodopsin (R*) to delineate the critical determinants of the G(t)α/R* coupling. In contrast to G(s)α, a chimeric G(s)α/G(t)α protein containing only 11 C-terminal residues from transducin was capable of binding to and being potently activated by R(*). Our results suggest that Cys347 and Gly348 are absolutely essential, whereas Asp346 is more modestly involved in the G(t) activation by R(*). In addition, the analysis of the intrinsic nucleotide exchange in mutant G(s)α indicated an interaction between the C terminus and the switch II region in G(t)α-GDP. Mutant G(s)α containing the G(t)α C terminus and substitutions of Asn239 and Asp240 (switch II) by the corresponding G(t)α residues, Glu212 and Gly213, displayed significant reductions in spontaneous guanosine 5'-O-(3- thiotriphosphate)-binding rates to the levels approaching those in G(t)α. Communication between the C terminus and switch II of G(t)α does not appear essential for the activational coupling between G(t) and R*, but may represent one of the mechanisms by which Gα subunits control intrinsic nucleotide exchange.