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 phosphodiester ase and the alpha subunit (T alpha) of transducin is a key step for th e regulation of cGMP phosphodiesterase in retinal rod outer segments. Here we have utilized a combination of specific modification by an end ogenous enzyme and site-directed mutagenesis of the P gamma polycation ic region to identify residues required for the interaction with T alp ha, P gamma, free or complexed with the alpha beta subunit (P alpha be ta) or cGMP phosphodiesterase, was specifically radiolabeled by prewas hed rod membranes in the presence of [adenylate-P-32]NAD. Identificati on of ADP-ribose in the radiolabeled P gamma and radiolabeling or argi nine-replaced mutant forms of P gamma indicate that both arginine 33 a nd arginine 36 are similarly ADP-ribosylated by endogenous ADP-ribosyl transferase, but only one arginine is modified at a time. P gamma comp lexed with T alpha (both GTP- and GDP-bound forms) was not ADP-ribosyl ated; however, agmatine, which cannot interact with T alpha, was ADP-r ibosylated int he presence of T alpha, suggesting that a P gamma domai n containing these arginines is masked by T alpha. A P gamma mutant (R 33,36K), as well as wild type P gamma, inhibited both GTP hydrolysis o f T alpha and GTP binding to T alpha. Moreover, GTP-bound T alpha acti vated P alpha beta that had been inhibited by R33,36K. However, anothe r 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 ha d been inhibited by R33,36L. These results indicate that a P gamma dom ain containing these arginines is required for its interaction with T alpha, but not with P alpha beta, and that positive charges in these a rginines are crucial for the interaction.