Abstract
Interaction between the gamma subunit (Pgamma) of cGMP phosphodiesterase and the alpha subunit (Talpha) 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 Pgamma polycationic region to identify residues required for the interaction with Talpha. Pgamma, free or complexed with the alphabeta subunit (Palphabeta) of cGMP phosphodiesterase, was specifically radiolabeled by prewashed rod membranes in the presence of [adenylate-32P]NAD. Identification of ADP-ribose in the radiolabeled Pgamma and radiolabeling of arginine-replaced mutant forms of Pgamma 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. Pgamma complexed with Talpha (both GTP- and GDP-bound forms) was not ADP-ribosylated; however, agmatine, which cannot interact with Talpha, was ADP-ribosylated in the presence of Talpha, suggesting that a Pgamma domain containing these arginines is masked by Talpha. A Pgamma mutant (R33,36K), as well as wild type Pgamma, inhibited both GTP hydrolysis of Talpha and GTP binding to Talpha. Moreover, GTP-bound Talpha activated Palphabeta that had been inhibited by R33,36K. However, another Pgamma mutant (R33,36L) could not inhibit these Talpha functions. In addition, GTP-bound Talpha could not activate Palphabeta inhibited by R33,36L. These results indicate that a Pgamma domain containing these arginines is required for its interaction with Talpha, but not with Palphabeta, and that positive charges in these arginines are crucial for the interaction.
Highlights
Interaction between the ␥ subunit (P␥) of cGMP phosphodiesterase and the ␣ subunit (T␣) of transducin is a key step for the regulation of cGMP phosphodiesterase in retinal rod outer segments
At least four different interactions are considered: (a) P␥-P␣ interaction for the inhibition of cGMP hydrolysis by P␣; (b) P␥-P␣ interaction for the stimulation of cGMP binding to P␣ noncatalytic sites; (c) P␥-T␣ interaction for the release of P␥ inhibitory strain from P␣; and (d) P␥-T␣ interaction for the release of P␥ to reduce the affinity of P␣ noncatalytic sites to cGMP
In this study we have focused on identification of specific residues in the P␥ polycationic region for following reasons. (i) The polycationic region has been suggested to be involved in the interaction with both P␣ and GTP1⁄7T␣ for the regulation of cGMP hydrolysis
Summary
Interaction between the ␥ subunit (P␥) of cGMP phosphodiesterase and the ␣ subunit (T␣) of transducin is a key step for the regulation of cGMP phosphodiesterase in retinal rod outer segments. In amphibian ROS, P␥ regulates these P␣ functions as an inhibitor of cGMP hydrolysis [12] and as a stimulator of cGMP binding to noncatalytic sites [13, 14]. Without impairing interaction with P␣, a frameshift mutation of P␥ has revealed that the carboxyl-terminal residues are involved in the cGMP hydrolysis inhibition [15]. The frameshift mutation of P␥ has suggested that the amino-terminal residues are involved in the stimulation of cGMP binding to noncatalytic sites on P␣ [15]. (i) The polycationic region has been suggested to be involved in the interaction with both P␣ and GTP1⁄7T␣ for the regulation of cGMP hydrolysis. We find that positive charges in these arginine are important for the interaction with T␣
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
