REGULATION OF THE CYCLIC GMP INHIBITED PHOSPHODIESTERASE

细胞内环磷酸腺苷（cAMP）浓度的增加抑制所有 血小板反应，包括形状改变、粘附、聚集和 分泌物 cAMP的水平在受体水平上受到控制 通过刺激或抑制腺苷酸环化酶或通过其水解 cAMP磷酸二酯酶（PDE）。 血小板中主要的PDE是 低Km cGMP抑制PDE（cGI PDE）。 cGI PDE的研究包括 正在进行中，涉及酶的克隆和表达， 利用分子生物学技术对活性位点区域进行研究。 先前 几个实验室的研究表明，这种酶是由 在不同于活性位点区域的位点磷酸化。 的 本提案的主要目的是界定 通过磷酸化血小板和HEL细胞中的cGI PDE，确定 磷酸化位点的氨基酸序列，并研究其结构- 功能关系。 第一个具体目标是比较 酶的非磷酸化和磷酸化形式的动力学 包括离体和体外。 第二个具体目标是确定 磷酸化位点的氨基酸序列。 的格局 将比较各种激酶的磷酸化。 最后的具体 目的是研究磷酸化的 通过定点诱变。 法规的特点 cGI PDE的表达可能会促进我们对细胞内信号传导的认识 机制等 这些见解可能对药物治疗有直接影响。 在选择性抑制血小板功能领域的发展。

Increased intracellular concentrations of cyclic AMP (cAMP) inhibit all platelet responses including shape change, adhesion, aggregation, and secretion. The levels of cAMP are controlled at the level of receptors by stimulation or inhibition of adenylate cyclase or by its hydrolysis by cAMP phosphodiesterases (PDEs). The predominant PDE in platelets is a low Km cGMP-inhibited PDE (cGI PDE). Studies of the cGI PDE are ongoing and involve cloning and expression of the enzyme in order to study the active site region by molecular biology techniques. Previous studies from several labs indicate that this enzyme is stimulated by phosphorylation at sites distinct from the active site region. The primary objective of this proposal is to delineate the regulation of the cGI PDE in platelets and HEL cells by phosphorylation, determine the amino acid sequence of the phosphorylated site and study the structure- function relationships. The first specific aim is to compare the kinetics of the nonphosphorylated and phosphorylated forms of the enzyme both ex vivo and in vitro. The second specific aim is to determine the amino acid sequence of the phosphorylation site (s). The pattern of phosphorylation by various kinases will be compared. The final specific aim is to study the structure-function relationship of the phosphorylated site by site-directed mutagenesis. Characterization of the regulation of the cGI PDE may advance our knowledge of intracellular cell signaling mechanisms. Such insights may have direct implications in drug development in the area of selective inhibition of platelet function.