Suppression of GTP/Tα-Dependent Activation of cGMP Phosphodiesterase by ADP-Ribosylation by Its γ Subunit in Amphibian Rod Photoreceptor Membranes

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Abstract
Our previous study has shown that Pγ, the regulatory subunit of cGMP phosphodiesterase (PDE), is ADP-ribosylated by endogenous ADP-ribosyltransferase when Pγ is free or complexed with the catalytic subunits of PDE in amphibian rod photoreceptor membranes. The Pγ domain containing ADP-ribosylated arginines was shown to be involved in its interaction with Tα, a key interaction for PDE activation. In this study, we describe a possible function of the Pγ ADP-ribosylation in the GTP/Tα-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 ∼50% of that of membranes preincubated without NAD. The Pγ release by the GTP-washing from these NAD-preincubated membranes was also suppressed to ∼50% of that preincubated without NAD. Taking into consideration that ∼50% of Pγ is ADP-ribosylated under these conditions, these observations suggest that the ADP-ribosylated Pγ cannot interact with GTP/Tα. We have also shown that a soluble fraction of ROS contains an enzyme(s) to release the radioactivity of [32P]ADP-ribosylated Pγ in concentration- and time-dependent manners, suggesting that the Pγ 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 Pγ, estimation of their molecular masses, ADP-ribosylation of Pγ arginine mutants, effects of ADP-ribosyltransferase inhibitors on the Pγ ADP-ribosylation, and effects of salts and pH on the Pγ ADP-ribosylation, indicates that rod ADP-ribosyltransferase contains two isozymes, and that these two isozymes have similar properties for the Pγ ADP-ribosylation. Our observations strongly suggest that the negative regulation of PDE through the reversible Pγ ADP-ribosylation may function in the phototransduction mechanism.

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