Rim and Exocytosis in Chromaffin Cells

描述(申请人提供)：激素和神经递质的胞吐释放是细胞间通讯和突触传递的中心，对内分泌、心血管和神经系统的正常功能至关重要。该应用的长期目标是了解调控胞吐的生化和生理机制。RIM已成为神经内分泌细胞和神经元胞吐的重要调节因子。它在胞吐作用中的作用将在这项提案中进行研究。RIM1是一种多结构域的Rab3a结合蛋白，位于神经末梢的活动区。基因敲除实验显示线虫和小鼠的突触传递存在缺陷。在嗜铬细胞分泌的生化检测中，RIM1及其同源家族成员RIM2强烈促进胞吐作用。指导这一提议的假设是，多模块蛋白RIM1和RIM2是整合胞吐关键蛋白功能的分子支架。一个基本的假设是，鉴定RIM中特定结构域与之相互作用的蛋白质并分析这些相互作用的功能后果不仅将解释RIM调节胞吐作用的机制，而且还将阐明这一复杂的、多步骤过程的协调的基本途径。RIM在胞吐途径中的功能将主要通过肾上腺嗜铬细胞和PC12细胞来确定。将使用生化和电生理技术。我们已经确定了在嗜铬细胞中表达时独立促进分泌的两个结构域，即锌指结构域和C2B结构域。我们已经确定了锌指结构域的两个看似合理的配体。这些蛋白质与锌指结构域的相互作用以及相互作用对分泌途径的影响将被研究。我们还将研究C2结构域的配体之间相互作用的功能意义，并寻找更多的配体。一个相关的问题，内源性Rim2在嗜铬细胞和PC12细胞中的功能也将被研究。用海马神经元培养的实验将研究Rim/Rab3a相互作用和RIM1 PDZ结构域在转基因RIM1在神经末梢定位中的作用。

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.