CHARACTERIZATION AND SORTING OF ZYMOGEN GRANULE PROTEINS

描述：外分泌胰腺是一个很好的研究系统 上皮极性、蛋白质分选和调节分泌。 这 应用建立在两个酶原颗粒的初始表征上 膜蛋白，VAMP和GP 2。 VAMP（囊泡相关膜蛋白）代表了一个完整的 对蛋白质的囊泡运输至关重要的膜蛋白。 最初的第一个奖项支持的研究确定， 与在突触囊泡中发现的VAMP-2类似， 胰酶原颗粒 然而，对VAMP的进一步研究表明， 胰腺中存在新的同种型。 一种新的VAMP的cDNA克隆 家族成员，目前命名为VAMP-3，已被分离， 序列确定。 这项建议的一个具体目的是， VAMP-3蛋白的亚细胞分布。 是否 VAMP-3的亚细胞分布与其他三种不同。 将测定先前表征的哺乳动物VAMP。 方法来 包括亚细胞分离、脉冲追踪研究， 显微镜 VAMP-3表现出一个大的囊泡内结构域， 已知的哺乳动物VAMP同种型。 这个唯一域的一个势函数 包括VAMP排序中的角色。 体外诱变将用于 产生具有人转铁蛋白受体的嵌合蛋白， VAMP-3独特结构域是否可以用作分选信号。 GP 2是酶原颗粒中的主要膜蛋白。 虽然其 功能目前尚不清楚，它可能是重要的外分泌 分泌物 最近的数据表明，GP 2在细胞内通过 在氨基末端的内切蛋白水解裂解。 基于这些假设 细胞内的加工过程调节GP 2的功能， 处理将被识别和突变。 这些GP 2加工突变体 然后将用于体内和体外研究以研究GP 2功能。 加工是否影响GP 2与自身或其他细胞结合的能力， 将测定酶原。 蛋白质加工在GP 2分选中的作用 也将被研究。 GP 2处理的表征将导致 更好地了解其功能，分类，以及在人类中的潜在作用 慢性胰腺炎等疾病。

DESCRIPTION: The exocrine pancreas represents an excellent system to study epithelial polarity, protein sorting, and regulated secretion. This application builds on the initial characterization of two zymogen granule membrane proteins, VAMP and GP2. VAMP (vesicle associated membrane protein) represents a family of integral membrane proteins that are essential to the vesicular transport of proteins. Studies supported by the initial First Award established that a protein similar to VAMP-2 found in synaptic vesicles is also present in the pancreatic zymogen granule. Further studies of VAMP, however, suggested a novel isoform is present in the pancreas. A cDNA clone for a new VAMP family member, currently named VAMP-3 has been isolated and the nucleotide sequence determined. A specific aim of this proposal is to characterize the subcellular the subcellular distribution of the VAMP-3 protein. Whether the subcellular distribution of VAMP-3 is distinct from the three other mammalian VAMPs previously characterized will be determined. Approaches to be used include subcellular fractionation, pulse chase studies, and microscopy. VAMP-3 exhibits a large intravesicular domain that differs from the other known mammalian VAMP isoforms. A potential function of this unique domain includes a role in VAMP sorting. In vitro mutagenesis will be used to generate chimeric proteins with the human transferrin receptor to determine whether the VAMP-3 unique domain can serve as a sorting signal. GP2 is the major membrane protein in the zymogen granule. Although its function is currently unclear, it is likely to be important in exocrine secretion. Recent data indicates that GP2 is processed intracellularly by endoproteolytic cleavage at the amino terminal end. Based on the hypotheses that intracellular processing regulates GP2 function, the sites for GP2 processing will be identified and mutated. These GP2 processing mutants will then be used in in vivo and in vitro studies to study GP2 function. Whether processing affects the ability of GP2 to bind to itself or to other zymogens will be determined. The role of protein processing in GP2 sorting will also be studied. Characterization of GP2 processing will lead to a better understanding of its function, sorting, and potential role in human diseases such as chronic pancreatitis.