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...