An effector site that stimulates G-protein GTPase in photoreceptors

Heterotrimeric G-proteins mediate between receptors and effectors, acting as molecular clocks. G-protein interactions with activated receptors catalyze the replacement of GDP bound to the α-subunit with GTP. α-Subunits then modulate the activity of downstream effectors until the bound GTP is hydrolyzed. In several signal transduction pathways, including the cGMP cascade of photoreceptor cells, the relatively slow GTPase activity of heterotrimeric G-proteins can be significantly accelerated when they are complexed with corresponding effectors. In the phototransduction cascade the GTPase activity of photoreceptor G-protein, transducin, is substantially accelerated in a complex with its effector, cGMP phosphodiesterase. Here we characterize the stimulation of transducin GTPase by a set of 23 mutant phosphodiesterase γ-subunits (PDE(γ)) 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 hydrolysis by transducin in a complex with this mutant. This mutation also resulted in a reduction of PDE(γ) affinity for transducin, but did not affect PDE(γ) interactions with the phosphodiesterase catalytic subunits. Single substitutions of 7 other hydrophobic amino acids resulted in a 50-70% reduction in the ability of PDE(γ) to stimulate transducin GTPase, while substitutions of charged and polar amino acids had little or no effect. These observations suggest that the role of PDE(γ) in activation of the transducin GTPase rate may be based on multiple hydrophobic interactions between these molecules.