Rim and Exocytosis in Chromaffin Cells

嗜铬细胞的边缘和胞吐作用

基本信息

批准号：
7086124
负责人：
RONALD W HOLZ
金额：
$ 26.63万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-05-14 至 2007-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7086124
关键词：
PC12 cells binding proteins chromaffin cells electrophysiology exocytosis hippocampus nerve endings neurotransmitter transport protein binding protein protein interaction protein structure function secretion tissue /cell culture transfection

项目摘要

DESCRIPTION (provided by applicant): Exocytotic release of hormones and neurotransmitters is central to intercellular communication and synaptic transmission and is fundamental to the normal function of the endocrine, cardiovascular and nervous systems. The long-term goal of the application is to understand the biochemical and physiological mechanisms underlying regulated exocytosis. Rim has emerged as an important regulator of exocytosis in neuroendocrine cells and neurons. Its function in exocytosis will be investigated in this proposal. Rim1 is a multidomain, Rab3a-binding protein that is located at active zones in nerve terminals. Gene knockout experiments reveal defects in synaptic transmission in C.elegans and mice. Rim1 and its closely homologous family member Rim2 strongly enhance exocytosis in biochemical assays of secretion in chromaffin cells. The hypothesis guiding this proposal is that the multi-modular proteins Rim1 and Rim2 are molecular scaffolds which integrate the functions of proteins critical for exocytosis. An underlying assumption is that identification of proteins with which specific domains in Rim interact and the analysis of the functional consequences of the interactions will not only explain the mechanisms through which Rim modulates exocytosis, but will also illuminate an essential pathway for coordination of this complex, multi-step process. Rim function in the exocytotic pathway will be determined using primarily adrenal chromaffin and PC12 cells. Biochemical and electrophysiological techniques will be used. We have identified two domains that independently enhance secretion when expressed in chromaffin cells, the Zn finger domain and the C2b domain. We have identified two plausible ligands for the Zn finger domain. The interaction of these proteins with the Zn finger domain and the consequences of the interactions on the secretory pathway will be investigated. We will also investigate the functional significance of the interaction of ligands of the C2 domains, which have been identified by others, and search for additional ligands. A related issue, the function of endogenous Rim2 in chromaffin and PC12 cells will also be investigated. Experiments with cultures of hippocampal neurons will investigate the roles of the Rim/Rab3a interaction and of the Rim1 PDZ domain in the localization of transfected Rim1 in nerve terminals.

描述（由申请人提供）：激素和神经递质的胞吐释放是细胞间通信和突触传播的核心，并且是内分泌，心血管和神经系统正常功能的基础。该应用的长期目标是了解受调节胞吐作用的生化和生理机制。 RIM已成为神经内分泌细胞和神经元中胞吐作用的重要调节剂。该提案将研究其在胞吞作用中的功能。 RIM1是一种多域结合蛋白，位于神经末端的活性区域。基因基因敲除实验揭示了甲状腺和小鼠的突触传播缺陷。 RIM1及其紧密同源的家族成员RIM2强烈增强了铬蛋白细胞分泌的生化测定中的胞吐作用。指导该提议的假设是多模块化蛋白RIM1和RIM2是分子支架，它们整合了对胞吐作用至关重要的蛋白质的功能。一个基本的假设是，鉴定蛋白质与RIM相互作用中特定结构域的蛋白质以及对相互作用的功能后果的分析不仅会解释RIM调节胞吐作用的机制，而且还将阐明该复合物多步过程的协调途径。胞吐途径中的边缘功能将使用主要是肾上腺染色体和PC12细胞确定。将使用生化和电生理技术。我们已经确定了两个域，这些结构域在铬蛋白细胞，锌指域和C2B结构域中独立增强分泌。我们已经确定了锌指域的两个合理的配体。这些蛋白质与锌指域的相互作用以及分泌途径上的相互作用的后果。我们还将研究C2结构域配体相互作用的功能意义，C2结构域已鉴定出来，并寻找其他配体。还将研究一个相关的问题，内源性RIM2在铬蛋白和PC12细胞中的功能也将被研究。海马神经元培养物的实验将研究RIM/RAB3A相互作用和RIM1 PDZ结构域在神经末端转染的RIM1定位中的作用。