Identification of Effector Residues on Photoreceptor G Protein, Transducin

Abstract

Transducin is a photoreceptor-specific heterotrimeric GTP-binding protein that plays a key role in the vertebrate visual transduction cascade. Here, using scanning site-directed mutagenesis of the chimeric G at/Gai1a-subunit(Gat/i), we identified Gat residues critical for interaction with the effector enzyme, rod cGMP phosphodiesterase (PDE). Our evidence suggests that residue Ile in the switch II region directly interacts with the effector in the active GTP-bound conformation of Gat. Residues Arg, Arg , and Trp are essential for the conformation-dependent Gat /effector interaction either via direct contacts with the inhibitory PDE g-subunit or by forming an effectorompetent conformation through the communication network between switch II and the switch III/a3-helixdomainofGa . Residues His 244 and Asn 247 in the a3helixofGat are responsible for the conformation-independent effector-specific interaction. Insertion of these residues rendered the G a t/i chimera with the ability to bind PDE g-subunitand stimulate PDE activity approaching that of native G at. Comparative analysis of the interactions of G a t/i mutants with PDE and RGS16 revealed two adjacent but distinct interfaces on transducin. This indicates a possibility for a functional trimeric complex, RGS/G a/effector, that may play a central role in turn-off mechanisms of G protein signaling systems, particularly in phototransduction.