Cyclic GMP phosphodiesterase, a key enzyme for phototransduction, contains ␣, ␤ (P␣␤), and two ␥ (P␥) subunits. In addition to catalytic sites, P␣␤ has two classes of noncatalytic cGMP binding sites with different affinities (K d values <100 nM and >1 M). P␥ regulates P␣␤ as an inhibitor of cGMP hydrolysis and as a stimulator of cGMP binding to the high affinity noncatalytic sites. P␥ release from P␣␤ by the GTP-bound ␣ subunit of transducin (GTP⅐T␣) interrupts these two functions. Here we describe a novel regulation of the P␥ release by [cGMP] and its physiological implication. We isolated P␥ mutants that exhibit abnormally one of these two functions, indicating the distinct domains in P␥ are involved to express these functions. When [cGMP] was high (ϳ5 M), P␥ responsible for the inhibition of cGMP hydrolysis was preferentially released, and cGMP hydrolysis activity of P␣␤ was increased about 10 times. When [cGMP] was low (less than ϳ0.5 M), P␥ responsible for the stimulation of cGMP binding to the high affinity sites was released. The P␥ release resulted in the decrease of relative affinity of cGMP for the high affinity sites to at least 1 ⁄10, followed by the rapid release of cGMP from one of the high affinity sites (apparent t1 ⁄2 ‫؍‬ 3.8 s). cGMP (ϳ5 M) inhibited the extraction of P␣␤ from rod membranes by a Mg 2؉ -free hypotonic buffer. The inhibition of P␣␤ extraction was not affected by P␥, suggesting that P␣␤ detects on the order of micromolar [cGMP] using low affinity noncatalytic sites on P␣␤. Because [cGMP] is ϳ5 M in darkness and lowered by photoexcitation and phosphodiesterase concentration is ϳ30 M in rod photoreceptors, it is possible that cGMP phosphodiesterase functions to increase cytoplasamic [cGMP] after [cGMP] is reduced to the illuminated level.Since Bitensky and Miller suggested the involvement of cyclic nucleotides in phototransduction (1), many investigators have contributed to establish the role of cGMP in phototransduction (1-3). The illuminated rhodopsin stimulates GTP/GDP exchange on T␣, 1 which in turn activates cGMP phosphodiesterase. The resulting decrease of cytoplasmic [cGMP] leads to closure of cGMP-gated channels and hyperpolarization of photoreceptors. The closure of channels also blocks Ca 2ϩ influx, while Ca 2ϩ efflux by a Na ϩ /Ca 2ϩ exchanger continues. The resulting decline in the free [Ca 2ϩ ] is believed to play a major role in the adaptation and recovery processes of photoreceptors by negative feedback regulation by [Ca 2ϩ ] (4). However, regulation of phototransduction by the decrease in cytoplasmic [cGMP] has never been clarified.Rod cGMP phosphodiesterase contains ␣, ␤ (P␣␤), and two ␥ (P␥) subunits. Previous studies have shown that P␣␤ has two catalytic sites for cGMP hydrolysis (5, 6) as well as two classes of noncatalytic cGMP binding sites with different affinities (K d values ϳ100 nM and 1-6 M) (5-9). These noncatalytic sites are the major cGMP binding sites in ROS, binding more than 90% of the cellular cGMP (7, 9). The roles of these noncatalytic s...

Interaction between the ␥ subunit (P␥) of cGMP phosphodiesterase and the ␣ subunit (T␣) of transducin is a key step for the regulation of cGMP phosphodiesterase in retinal rod outer segments. Here we have utilized a combination of specific modification by an endogenous enzyme and site-directed mutagenesis of the P␥ polycationic region to identify residues required for the interaction with T␣. P␥, free or complexed with the ␣␤ subunit (P␣␤) of cGMP phosphodiesterase, was specifically radiolabeled by prewashed rod membranes in the presence of [adenylate-32 P]NAD. Identification of ADP-ribose in the radiolabeled P␥ and radiolabeling of arginine-replaced mutant forms of P␥ indicate that both arginine 33 and arginine 36 are similarly ADP-ribosylated by endogenous ADP-ribosyltransferase, but only one arginine is modified at a time. P␥ complexed with T␣ (both GTP-and GDP-bound forms) was not ADP-ribosylated; however, agmatine, which cannot interact with T␣, was ADP-ribosylated in the presence of T␣, suggesting that a P␥ domain containing these arginines is masked by T␣. A P␥ mutant (R33,36K), as well as wild type P␥, inhibited both GTP hydrolysis of T␣ and GTP binding to T␣. Moreover, GTP-bound T␣ activated P␣␤ that had been inhibited by R33,36K. However, another P␥ mutant (R33,36L) could not inhibit these T␣ functions. In addition, GTP-bound T␣ could not activate P␣␤ inhibited by R33,36L. These results indicate that a P␥ domain containing these arginines is required for its interaction with T␣, but not with P␣␤, and that positive charges in these arginines are crucial for the interaction.Cyclic GMP phosphodiesterase (PDE), 1 a key enzyme in phototransduction, is composed of P␣␤ and two P␥ subunits (1-6). P␣␤ hydrolyzes cGMP (7,8) and binds cGMP to its high affinity, noncatalytic sites (9 -11). In amphibian ROS, P␥ regulates these P␣␤ functions as an inhibitor of cGMP hydrolysis (12) and as a stimulator of cGMP binding to noncatalytic sites (13,14). Different interactions between P␣␤ and P␥ have been suggested to be required to express these two functions (15, 16). In bovine ROS, P␥ inhibits cGMP hydrolysis by P␣␤ (17); however, the effect of P␥ on the cGMP binding to noncatalytic sites has never been documented. In amphibian ROS, these P␥ functions are interrupted by P␥ release with GTP⅐T␣ from P␣␤ (12-14, 18). We have recently suggested that these functionally different P␥s are released in the different steps of phototransduction (15, 16). When [cGMP] is at the dark level, P␥ responsible for the inhibition of cGMP hydrolysis is released. Consequently, cGMP is hydrolyzed by the activated PDE for photoexcitation. When [cGMP] becomes low, P␥ responsible for the stimulation of cGMP binding is released, and the affinities of these noncatalytic sites to cGMP are drastically reduced. The resulting release of cGMP from these noncatalytic sites may facilitate the recovery of cytoplasmic [cGMP] to the dark level in ROS.To understand physiological functions of protein-protein interaction, functional structures of the protein i...