CGMP-BINDING PHOSPHODIESTERASE--REGULATORY MECHANISMS

像cAMP一样，cGMP现在被确定为重要的第二信使， 调节各种生理过程。 相对于 在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依赖性蛋白激酶 cGMP门控离子通道，可能还有cAMP依赖性蛋白激酶 通过cGMP的“交叉激活”。 这一长期目标 研究将是确定作用机制和细胞 cGMP结合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，视觉磷酸二酯酶是 在这个级联反应的酶。cGMP也可能参与神经 记忆等功能。cGMP也介导平滑肌松弛 由激动剂如心房利钠肽、一氧化氮和 可能是新发现的鸟苷肽的作用。 治疗 或通过cGMP起作用的病理性药剂包括硝基血管扩张剂 (e.g.,硝酸甘油），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 通常用于缓解胸痛，哮喘，男性阳痿，高 血压. cGMP-结合cGMP-特异性磷酸二酯酶将在COS-1中过表达。 cells，E. coli或SF 9/杆状病毒。 定点突变和 合成肽将用于研究亮氨酸拉链基序， 提供酶的二聚化。 使用天然和重组 酶，最近发现的Zn 2 +-结合组分，保守在 磷酸二酯酶催化结构域，将研究使用原子 吸收光谱法、65 Zn 2+结合、诱变和合成 缩氨酸将对cGMP结合位点进行定点突变 以确定cGMP结合的元件和功能。 对cGMP结合和催化位点特异的类似物也将用于 研究结合位点功能。 磷酸二酯酶的功能变化 将在蛋白激酶磷酸化后和 通过磷蛋白磷酸酶去磷酸化。 的磷酸化 磷酸二酯酶也将通过研究32 p掺入 这种酶在完整的细胞中。

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.