SECRETORY GRANULE MEMBRANE IN REGULATED EXOCYTOSIS

这项提案寻求在未来五年内界定 分泌颗粒膜对外分泌的调节作用。这个 颗粒膜隔室包含一套独特的成分 不同外分泌组织共有的相关蛋白质。很可能是 其中一些蛋白质在功能上参与调节分泌。 事实上，几名研究人员对神经体液的鉴定- 受调节的分泌颗粒膜磷蛋白支持这一点 假设。此外，最近的证据表明， 特定颗粒膜蛋白和酶的磷酸化 分泌物。这个项目旨在描述，在利率模型中， 分泌颗粒膜磷蛋白在调节中的作用 外分泌的物质。从该项目获得的信息可能 有助于我们对临床相关疾病的理解 胞吐，如囊性纤维化和实验性胰腺炎。 对此有四个具体目标和方法论途径 求婚。第一，磷酸化与蛋白水解酶活性之间的关系 完整细胞中的特定颗粒膜蛋白和分泌物将 用抗外分泌分泌颗粒的抗血清检测 膜蛋白。调控蛋白之间的密切相关性 磷酸化和酶分泌将有助于识别 用于后续分析的具有重要生理意义的磷蛋白。 第二，将使用快速颗粒纯化程序来确认 这些磷蛋白定位于其中一种分泌物 车厢。第三，磷蛋白将在结构上 以确定它们在分泌物中的保守程度为特征的 系统。第四，将采用体外磷酸化技术来 将给定的第二信使通路指定为特定的磷酸化 事件。最后，单特异性多克隆抗体将被提高到 用于确定精确亚细胞的特定磷酸蛋白 这些物种的定位以及制备亲和层析柱 大量氨基酸所需蛋白质的纯化 测序。

This proposal seeks, over the next five years, to define the role of the secretory granule membrane in the regulation of exocrine secretion. The granule membrane compartment contains a unique set of compositionally related proteins common to different exocrine tissues. It is likely that some of these proteins are functionally involved in regulating secretion. Indeed, the identification by several investigators of neurohumorally- regulated secretory granule membrane phosphoproteins supports this hypothesis. Further, recent evidence shows a close correlation between the phosphorylation of specific granule membrane proteins and enzyme secretion. This project is designed to characterize, in the rate model, the role of secretory granule membrane phosphoproteins in the regulation of exocrine secretion. Information obtained from this project may contribute to our understanding of clinically-related disorders of exocytosis such as cystic fibrosis and experimental pancreatitis. There are four specific aims and methodological approaches to this proposal. First, the relationship between the phosphorylation of specific granule membrane proteins and secretion in intact cells will be examined using an antiserum raised against exocrine secretory granule membrane proteins. A close correlation between regulated protein phosphorylation and enzyme secretion will serve to identify physiologically important phosphoproteins for subsequent analysis. Second, a rapid granule purification procedure will be used to confirm localization of these phosphoproteins to one of the secretory compartments. Third, the phosphoproteins will be structurally characterized to determine their degree of conservation among secretory systems. Fourth, in vitro phosphorylation techniques will be employed to assign given second messenger pathways to specific phosphorylation events. Lastly, monospecific polyclonal antibodies will be raised to specific phosphoproteins for use in determining the precise subcellular localization of these species as well as to prepare affinity columns for the purification of large enough quantities of protein for amino acid sequencing.