REGULATION OF ROD CYCLIC GMP PHOSPHODIESTERASE

环鸟苷酸磷酸二酯酶（cyclicGMP phosphodiesterase，PDE）是脊椎动物的效应酶 调节第二信使水平的感光细胞， 环GMP。 PDE由催化性Pabeta亚基和两个抑制性Pabeta亚基组成。 P γ亚基。 Pgamma亚基使酶的活性保持在低水平， 黑了 光激活感光细胞后，GTP结合的α- 视杆细胞G蛋白转导素的亚基与PDE相互作用并激活PDE。 为了更好地理解转导蛋白激活PDE的机制， 了解P γ如何相互作用的结构细节是非常重要的， 并抑制pDE催化亚基。 当帕尔法贝塔的遗址- Pgamma相互作用主要定位于Pgamma亚基上，目前还不清楚 关于PDE催化亚基上这种相互作用的位点。 还有， 特定氨基酸残基在相互作用中的作用， 抑制Palphabeta和Pgamma的一级结构 对多肽的了解很少。 在本提案中， 参与Palpha（beta）-Pgamma的氨基酸残基 两种蛋白质的相互作用将使用交联，合成 肽图谱研究和Palpha（β）的定点诱变， Pgamma 此外，非催化性cGMP结合位点对 Palphabeta对PDE功能的影响和Pgamma对PDE活性的抑制作用将 追究 这些研究将有助于阐明 G蛋白的效应子激活。 他们还将提供一个深入了解 调节环核苷酸活性的一般机制 磷酸二酯酶，这可能导致设计新的药物， 控制细胞中的环核苷酸水平。 事实上，有些形式 视网膜变性的发生可能与PDE突变有关 强调了拟议研究的临床重要性。

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.