AN EFFECTOR SITE THAT STIMULATES G-PROTEIN GTPASE IN PHOTORECEPTORS

Heterotrimeric G-proteins mediate between receptors and effecters, act ing as molecular clocks. G-protein interactions with activated recepto rs catalyze the replacement of GDP bound to the alpha-subunit with GTP . alpha-Subunits then modulate the activity of downstream effecters un til the bound GTP is hydrolyzed. In several signal transduction pathwa ys, including the cGMP cascade of photorecepter cells, the relatively slow GTPase activity of heterotrimeric G-proteins can be significantly accelerated when they are complexed with correspending effecters. In the phototransduction cascade the GTPase activity of photoreceptor G-p rotein, transducin, is substantially accelerated in a complex with its effector, cGMP phosphodiesterase. Here we characterize the stimulatio n of transducin GTPase by a set of 23 mutant phosphodiesterase gamma-s ubunits (PDE(gamma)) containing single alanine substitutions within a stretch of the 25 C-terminal amino acid residues known to be primarily responsible for the GTPase regulation. The substitution of tryptophan at position 70 completely abolished the acceleration of GTP hydrolysi s by transducin in a complex with this mutant. This mutation also resu lted in a reduction of PDE(gamma) affinity for transducin, but did not affect PDE(gamma) interactions with the phosphodiesterase catalytic s ubunits. Single substitutions of 7 other hydrophobic amino acids resul ted in a 50-70% reduction in the ability of PDE(gamma) to stimulate tr ansducin GTPase, while substitutions of charged and polar amino acids had little or no effect. These observations suggest that the role of P DE(gamma) in activation of the transducin GTPase rate may be based on multiple hydrophobic interactions between these molecules.