SECRETORY GRANULE MEMBRANE IN REGULATED EXOCYTOSIS

该提案旨在在今后五年内确定 分泌颗粒膜参与外分泌的调节。 的 颗粒膜隔室包含一组独特的成分， 不同外分泌组织共有的相关蛋白质。 很可能 这些蛋白质中的一些在功能上参与调节分泌。 事实上，几个研究人员对神经体液的鉴定- 调节分泌颗粒膜磷蛋白支持这一点 假说. 此外，最近的证据表明， 特定颗粒膜蛋白和酶磷酸化 分泌物 该项目旨在描述，在速率模型中， 分泌颗粒膜磷蛋白在调节 外分泌的分泌物。 从本项目获得的信息可能 有助于我们理解临床相关疾病， 胞吐作用，如囊性纤维化和实验性胰腺炎。 这方面有四个具体目标和方法 提议 第一，磷酸化之间的关系 完整细胞中的特异性颗粒膜蛋白和分泌物将被 用抗外分泌颗粒的抗血清检测 膜蛋白 调节蛋白之间的密切相关性 磷酸化和酶分泌将用于识别 生理上重要的磷蛋白用于随后的分析。 其次，将使用快速颗粒纯化程序来确认 这些磷蛋白定位到一个分泌的 隔间 第三，磷蛋白将在结构上 其特征在于确定它们在分泌型之间的保守程度， 系统. 第四，将采用体外磷酸化技术， 将给定的第二信使途径分配给特定的磷酸化 事件 最后，将产生单特异性多克隆抗体， 用于测定精确亚细胞 这些物种的定位以及制备亲和柱， 纯化足够量蛋白质用于氨基酸 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.