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

The C-terminal regions of the heterotrimeric Gr protein ru-subunits play ke y roles in selective activation of G proteins by their cognate receptors, I n this study, mutant G(S)alpha proteins with substitutions by C-terminal re sidues of transducin (G(t)alpha) mere analyzed for their interaction with l ight-activated rhodopsin (R*) to delineate the critical determinants of the G(t)alpha/R* coupling, In contrast to G(S)alpha, a chimeric G(S)alpha/G(t) alpha protein containing only 11 C-terminal residues from transducin was ca pable of binding to and being potently activated by R*, Our results suggest that Gys(347) and Gly(348) are absolutely essential, whereas Asp(346) is m ore modestly involved in the G(t) activation by R*. In addition, the analys is of the intrinsic nucleotide exchange in mutant G(S)alpha indicated an in teraction between the C terminus and the switch II region in G(t)alpha.GDP. Mutant G(s)alpha containing the G(t)alpha C terminus and substitutions of Asn(239) and Asp(240) (switch II) by the corresponding C(t)alpha residues, Glu(212) and Gly(213), displayed significant reductions in spontaneous guan osine 5'-O-(3-thiotriphosphate)-binding rates to the levels approaching tho se in G,cu, Communication between the C terminus and switch II of G(t)alpha does not appear essential for the activational coupling between G, and R*, but may represent one of the mechanisms by which G alpha subunits control intrinsic nucleotide exchange.