CGMP-BINDING PHOSPHODIESTERASE--REGULATORY MECHANISMS

像cAMP一样，cGMP现在被确立为一个重要的第二信使， 调节多种生理过程。与之相对的是 CAMP系统中，哺乳动物细胞中存在多种cGMP受体。这些 包括cGMP结合磷酸二酯酶、cGMP依赖的蛋白激酶 CGMP门控离子通道，可能还有cAMP依赖的蛋白激酶 通过cGMP的“交叉激活”。这样做的长期目标是 调查将确定其作用机制和细胞 CGMP结合的cGMP特异性磷酸二酯酶的调节。这种酶 与视觉系统的磷酸二酯酶密切相关。CGMP是 视觉的第二信使，视觉的磷酸二酯酶是 在这个级联反应酶中。CGMP可能也参与了神经细胞 功能，如内存。CGMP也介导了平滑肌的松弛 由心钠素、一氧化氮等激动剂引起 可能是新发现的鸟苷肽的影响。治疗性的 或通过cGMP发挥作用的病理性药物包括硝基血管扩张剂 (例如，硝化甘油)、甲基黄嘌呤(例如，咖啡因)和一些 引起分泌性腹泻的肠毒素。升高cGMP的药物有 常用于缓解胸痛、哮喘、男性阳萎和性欲亢奋。 血压。 CGMP结合的cGMP特异性磷酸二酯酶将在COS- 细胞、大肠杆菌或SF9/杆状病毒。定点突变和一种 合成肽将用于研究一种亮氨酸拉链基序，该基序可能 为酶的二聚化提供条件。使用原生和重组 酶是一种新近发现的与锌结合的成分，它在 磷酸二酯酶催化结构域，将用原子进行研究 吸收光谱、~(65)锌结合、诱变和合成 多肽。定点突变将在cGMP结合位点上进行 以确定cGMP结合的元件和功能。 针对cGMP结合和催化位点的类似物也将用于 研究结合位点的功能。磷酸二酯酶的功能变化 将在蛋白激酶磷酸化后和之后进行测量 通过磷酸化蛋白磷酸酶去磷酸化。蛋白的磷酸化 还将通过研究~(32)P掺入 这种酶在完好的细胞中。

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.