Residues within the polycationic region of cGMP phosphodiesterase gamma subunit crucial for the interaction with transducin alpha subunit - Identification by endogenous ADP-ribosylation and site-directed mutagenesis

Interaction between the gamma subunit (P gamma) of cGMP phosphodiesterase and the alpha subunit (T alpha) 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 gamma polycationic region to identify residues required for the interaction with T alpha, P gamma, free or complexed with the alpha beta subunit (P alpha beta) or cGMP phosphodiesterase, was specifically radiolabeled by prewashed rod membranes in the presence of [adenylate-P-32]NAD. Identification of ADP-ribose in the radiolabeled P gamma and radiolabeling or arginine-replaced mutant forms of P gamma 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 gamma complexed with T alpha (both GTP- and GDP-bound forms) was not ADP-ribosylated; however, agmatine, which cannot interact with T alpha, was ADP-ribosylated int he presence of T alpha, suggesting that a P gamma domain containing these arginines is masked by T alpha. A P gamma mutant (R33,36K), as well as wild type P gamma, inhibited both GTP hydrolysis of T alpha and GTP binding to T alpha. Moreover, GTP-bound T alpha activated P alpha beta that had been inhibited by R33,36K. However, another P gamma mutant (R33,36L) could not inhibit these T alpha functions. In addition, GTP-bound T alpha could not activate P alpha beta that had been inhibited by R33,36L. These results indicate that a P gamma domain containing these arginines is required for its interaction with T alpha, but not with P alpha beta, and that positive charges in these arginines are crucial for the interaction.