NONCATALYTIC CGMP-BINDING SITES OF AMPHIBIAN ROD CGMP PHOSPHODIESTERASE CONTROL INTERACTION WITH ITS INHIBITORY GAMMA-SUBUNITS - A PUTATIVE REGULATORY MECHANISM OF THE ROD PHOTORESPONSE

The cGMP phosphodiesterase (PDE) of retinal rods plays a central role in phototransduction. Illumination leads to its activation by a rod G-protein (G(t), transducin), thus causing a decrease in intracellular cGMP concentration, closure of plasma membrane cationic channels gated by cGMP, and development of the photoresponse. The PDE holoenzyme is an alphabetagamma2 tetramer. The alpha- and beta-subunits each contain one catalytic and one, or possibly two, noncatalytic cGMP-binding sites. Two identical gamma-subunits serve as protein inhibitors of the enzyme. Their inhibition is removed when they bind to G(t)-GTP during PDE activation. Here we report that the noncatalytic cGMP-binding sites regulate the binding of PDE(alphabeta) with PDE(gamma) and as a result determine the mechanism of PDE activation by G(t). If the noncatalytic sites are empty, G(t)-GTP physically removes PDE(gamma) from PDE(alphabeta) upon activation. Alternatively, if the noncatalytic sites are occupied by cGMP, G(t)-GTP releases PDE(gamma) inhibitory action but remains bound in a complex with the PDE heterotetramer. The kinetic parameters of activated PDE in these two cases are indistinguishable. This mechanism appears to have two implications for the physiology of photoreceptor cells. First, the tight binding of PDE(gamma) with PDE(alphabeta) when the noncatalytic sites are occupied by cGMP may be responsible for the low level of basal PDE activity observed in dark-adapted cells. Second, occupancy of the noncatalytic sites ultimately controls the rate of PDE inactivation (cf. Arshavsky, V. Yu., and Bownds, M. D. (1992) Nature 357, 416-417), for the GTPase activity that terminates PDE activity is slower when these sites are occupied and G(t) stays in a complex with PDE holoenzyme. In contrast GTPase acceleration is maximal when the noncatalytic sites are empty and G(t)-PDE(gamma) dissociates from PDE(alphabeta). Because cGMP levels are known to decrease upon illumination over a concentration range corresponding to the binding constants of the noncatalytic sites, the binding might be involved in determining the lifetime of activated PDE, after a single flash and/or during dark adaptation.