Like cAMP, cGMP is now established as an important second messenger that modulates a wide variety of physiological processes. In contrast to the cAMP system, there are multiple cGMP receptors in mammalian cells. These include cGMP-binding phosphodiesterases, cGMP-dependent protein kinases, cGMP-gated ion channels, and perhaps cAMP-dependent protein kinases through """"""""cross-activation"""""""" by cGMP. The long term objective of this investigation will be to determine the mechanism of action and cellular regulation of a cGMP-binding cGMP-specific phosphodiesterase. This enzyme is closely related to the phosphodiesterases of the visual system. cGMP is the second messenger for vision, and the visual phosphodiesterase is the responsive enzyme in this cascade. cGMP may also be involved in neural functions such as memory. cGMP also mediates smooth muscle relaxation caused by agonists such as atrial natriuretic peptide, nitric oxide, and possibly effects of the newly discovered guanylin peptides. Therapeutic or pathological agents that act through cGMP include nitrovasodilators (e.g., nitroglycerin), methylxanthines (e.g., caffeine), and some enterotoxins that cause secretory diarrhea. Agents that elevate cGMP are commonly used for relief of chest pain, asthma, male impotence, and high blood pressure. cGMP-binding cGMP-specific phosphodiesterase will be overexpressed in COS- cells, E. coli, or SF9/baculovirus. Site-directed mutagenesis and a synthetic peptide will be used to study a leucine zipper motif that may provide for dimerization of the enzyme. Using native and recombinant enzyme, a recently discovered Zn2+-binding component, conserved in phosphodiesterase catalytic domains, will be studied using atomic absorption spectrometry, 65Zn2+ binding, mutagenesis, and synthetic peptides. Site-directed mutagenesis will be done on the cGMP-binding sites of the enzyme to determine elements and function for cGMP binding. Analogs specific for cGMP binding and catalytic sites will also be used to study binding site functions. Functional changes of the phosphodiesterase will be measured after phosphorylation by protein kinases and after dephosphorylation by phosphoprotein phosphatases. Phosphorylation of the phosphodiesterase will also be examined by studying 32p incorporation into this enzyme in intact cells.
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