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
Va. Bondarenko et al., 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, The Journal of biological chemistry, 272(25), 1997, pp. 15856-15864
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.