Cloning of frog rod photoreceptor cGMP phosphodiesterase subunits (P-alpha, P-beta, P-gamma) and identification of P-gamma) sites involved in the stimulation of cGMP binding to P-alpha and P-beta non-catalytic sites

Retinal cGMP phosphodiesterase (PDE6) is a key enzyme in vertebrate phototransduction. Rod PDE contains two homologous catalytic subunits (Pd two identical regulatory subunits (P). Biochemical studies have shown that amphibian P has high affinity, cGMP-specific, non-catalytic binding sites and that P stimulates cGMP binding to these sites. Here we show by molecular cloning that each catalytic subunit in amphibian PDE, as in its mammalian counterpart, contains two homologous tandem GAF domains in its N-terminal region. In P-depleted membrane-bound PDE (20-40% P still present), a single type of cGMP-binding site with a relatively low affinity (Kd 100 nM) was observed, and addition of P increased both the affinity for cGMP and the level of cGMP binding. We also show that mutations of amino acid residues in four different sites in P reduced its ability to stimulate cGMP binding. Among these, the site involved in P phosphorylation by Cdk5 (positions 20-23) had the largest effect on cGMP binding. However, except for the C terminus, these sites were not involved in P inhibition of the cGMP hydrolytic activity of P. In addition, the P concentration required for 50% stimulation of cGMP binding was much greater than that required for 50% inhibition of cGMP hydrolysis. These results suggest that the P heterodimer contains two spatially and functionally distinct types of P-binding sites: one for inhibition of cGMP hydrolytic activity and the second for activation of cGMP binding to GAF domains. We propose a model for the P-P interaction in which P, by binding to one of the two sites in P, may preferentially act either as an inhibitor of catalytic activity or as an activator of cGMP binding to GAF domains in frog PDE.