Suppression of GTP/T alpha-dependent activation of cGMP phosphodiesterase by ADP-ribosylation by its gamma subunit in amphibian rod photoreceptor membranes.
Our previous study has shown that P gamma, the regulatory subunit of cGMP phosphodiesterase (PDE), is ADP-ribosylated by endogenous ADP-ribosyltransferase when P gamma is free or complexed with the catalytic subunits of PDE in amphibian rod photoreceptor membranes. The P gamma domain containing ADP-ribosylated arginines was shown to be involved in its interaction with T alpha, a key interaction for PDE activation. In this study, we describe a possible function of the P gamma ADP-ribosylation in the GTP/T alpha-dependent PDE activation. When rod membranes were preincubated with or without NAD and washed with a buffer containing GTP, the PDE activity of NAD-preincubated membranes was increased by the GTP-washing only to approximately 50% of that of membranes preincubated without NAD. The P gamma release by the GTP-washing from these NAD-preincubated membranes was also suppressed to approximately 50% of that preincubated without NAD. Taking into consideration that approximately 50% of P gamma is ADP-ribosylated under these conditions, these observations suggest that the ADP-ribosylated P gamma cannot interact with GTP/T alpha. We have also shown that a soluble fraction of ROS contains an enzyme(s) to release the radioactivity of [32P]ADP-ribosylated P gamma in concentration- and time-dependent manners, suggesting that the P gamma ADP-ribosylation is reversible. Rod ADP-ribosyltransferase solubilized from membranes by phosphatidylinositol-specific phospholipase C was separated into two fractions by ion-exchange columns. Biochemical characterization of these two fractions, including measurement of the Km for NAD and P gamma, estimation of their molecular masses, ADP-ribosylation of P gamma arginine mutants, effects of ADP-ribosyltransferase inhibitors on the P gamma ADP-ribosylation, and effects of salts and pH on the P gamma ADP-ribosylation, indicates that rod ADP-ribosyltransferase contains two isozymes, and that these two isozymes have similar properties for the P gamma ADP-ribosylation. Our observations strongly suggest that the negative regulation of PDE through the reversible P gamma ADP-ribosylation may function in the phototransduction mechanism.
Bondarenko
V A
VA
Kresge Eye Institute, Department of Ophthalmology, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
Yamazaki
M
M
Hayashi
F
F
Yamazaki
A
A
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D004789:Enzyme Activation / Q000187:drug effects; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008566:Membranes / Q000201:enzymology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000378:metabolism; D011892:Rana catesbeiana; D012374:Rod Cell Outer Segment / Q000201:enzymology* / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D015293:Transducin / Q000502:physiology*; D010738:Type C Phospholipases / Q000378:metabolism
D007527:Isoenzymes; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D000246:Adenosine Diphosphate Ribose; D012965:Sodium Chloride; D006160:Guanosine Triphosphate; D011065:Poly(ADP-ribose) Polymerases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; C046422:ADP-ribosylarginine hydrolase; D015293:Transducin; D043265:Phosphatidylinositol Diacylglycerol-Lyase