Cyclic GMP phosphodiesterase (PDE) is the effector enzyme of vertebrate photoreceptor cells which regulates the level of the second messenger, cyclic GMP. PDE consists of catalytic Pabeta subunits, and two inhibitory Pgamma subunits. The Pgamma subunits keep the enzyme's activity low in the dark. Upon light-activation of photoreceptor cells, the GTP-bound alpha- subunit of the rod G-protein transducin interacts with and activates PDE. In order to better understand the mechanism of PDE activation by transducin it is very important to know the structural details of how Pgamma interacts with and inhibits the pDE catalytic subunits. While sites of Palphabeta- Pgamma interaction have been located on Pgamma subunit, little is known about the sites of this interaction on the PDE catalytic subunit. Also, the role of the specific amino-acid residues for the interaction and inhibition on the primary structure of the both Palphabeta and Pgamma polypeptides is poorly understood. In this proposal, sites and specific amino-acid residues that are involved in the Palpha(beta)-Pgamma interaction on both proteins will be studied using cross-linking, synthetic peptide mapping studies and site-directed mutagenesis of Palpha(beta) and Pgamma. In addition, the role of non-catalytic cGMP binding sites on Palphabeta for PDE function and inhibition of PDE activity by Pgamma will be investigated. These studies will help to elucidate the basic molecular mechanisms of effector activation by G-proteins. They will also provide an insight into general mechanisms of regulation of activity of cyclic nucleotides phosphodiesterases which may lead to the design of new drugs directed to the control of cyclic nucleotides level in cells. The fact that some forms of retinal degeneration are likely to be connected to mutations of PDE underlines the clinical importance of the proposed study.
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