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

Bondarenko, V.A.; Desai, M.; Dua, S.; Yamazaki, M.; Amin, R.H.; Yousif, K.K.; Kinumi, T.; Ohashi, M.; Komori, N.; Matsumoto, H.; Jackson, K.W.; Hayashi, F.; Usukura, J.; Lipkin, V.M.; Yamazaki, A.

Journal of Biological Chemistry 272(25): 15856-15864

1997


ISSN/ISBN: 0021-9258
PMID: 9188484
Accession: 009344421

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Abstract
Interaction between the gamma subunit (P-gamma) of cGMP phosphodiesterase and the a 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) of cGMP phosphodiesterase, was specifically radiolabeled by prewashed rod membranes in the presence of (adenylate-32P)NAD. Identification of ADP-ribose in the radiolabeled P-gamma and radiolabeling of 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 in the 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 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.