| Interest in mammalian cGMP cascades has recently been intense. Much research in this area has centered on the signal receptors and guanylyl cyclases that modulate cGMP formation, as well as on cellular actions of cGMP. Natural signals (natriuretic peptides, guanylins and nitric oxide), in addition to many medications, stimulate cGMP cascades by activating these cyclases. Cyclic nucleotide phosphodiesterases (PDEs), which are also highly modulated enzymes, participate in regulating cellular cAMP and cGMP levels by catalyzing breakdown of cAMP and cGMP. The subject of this application is the cGMP-binding cGMP-specific PDE (PDE5), which is specific for cGMP over cAMP at its allosteric cGMP-binding sites and at its catalytic site. The long-term goal is to determine the mechanism of action and cellular regulation of PDE5, a major determinant of cGMP level in many tissues. Classical effects of cGMP include relaxation of smooth muscle, inhibition of platelet activation, neutrophil degranulation, and mediation of vision. New discoveries have expanded this list to include regulation of gene expression, chloride transport in intestine and kidney, watertransport, bone resorption, melanogenesis in skin, long-term nerve depression, and opioid effects. Therapeutic agents that elevate cGMP include PDE inhibitors (e.g., caffeine, papaverine, and sildenafil) and nitrovasodilators (e.g., nitroglycerin). Certain enterotoxins cause secretory diarrhea by elevating cGMP. Dr. Corbin recently demonstrated that in some instances cGMP acts by "crossactivating" cAMP receptors. However, PDE5 is a specific intracellular receptor for cGMP. The present proposal represents a thorough biochemical and physiological investigation of PDE5 regulation. This enzyme is the specific target of sildenafil (Viagra.), which is used in treatment of male erectile dysfunction associated with diabetes, aging, spinal cord injuries and other pathologies. Effects of phosphorylation of PDE5 by cyclic nucleotide-dependent protein kinases on catalysis and allosteric cGMP binding will be explored, and effects of mutating the phosphorylation site, Ser- 102 (human), to Ala, Glu, and Asp will be examined. The principal investigator will study the PDE5 regulatory domain structure and function by expressing truncation mutants that include various combinations of the two allosteric cUMP-binding sites and the phosphorylation site of the enzyme but exclude the catalytic domain. Small angle x-ray scattering and Fourier transformed infra-red spectroscopy will be used to study effects of cGMP and phosphorylation on conformation of these domains. Crystallography of certain truncated regulatory domains will be attempted. The specificity and efficacy with which phosphoprotein phosphatases of vascular smooth muscle extracts catalyze dephosphorylation of Ser-102 will be investigated. Short-term regulation of PDE5 in intact vascular smooth muscle by cGMP analogs, phosphoprotein phosphatase inhibitors, and agents that elevate cGMP will be studied. The possibility that these agents could alter enzyme activity, subcellular localization, or cGMP levels (feedback mechanism) will be examined. Potential non-covalent modulators of PDE5 other than cGMP will be sought. The possibility that PDE5 allosteric sites represent a sequestration site for cGMP in cells will be explored. Results of these studies will provide a basis for understanding fundamental questions relating to cGMP signaling in many tissues. |
