Rim and Exocytosis in Chromaffin Cells

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讯和突触传递的核心，是内分泌、心血管和神经系统正常功能的基础。该应用程序的长期目标是了解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胞吐作用的重要调节因子。它在胞吐作用中的功能将在本建议中进行研究。Rim 1是一种多结构域的Rab 3a结合蛋白，位于神经末梢的活性区。基因敲除实验揭示了秀丽隐杆线虫和小鼠的突触传递缺陷。在嗜铬细胞分泌的生化测定中，Rim 1及其密切同源的家族成员Rim 2强烈增强胞吐作用。指导这一提议的假设是，多模块蛋白质Rim 1和Rim 2是整合了胞吐作用关键蛋白质功能的分子支架。一个基本的假设是，识别与Rim中的特定结构域相互作用的蛋白质，并分析相互作用的功能后果，不仅可以解释Rim调节胞吐作用的机制，而且还可以阐明协调这一复杂的多步骤过程的重要途径。将主要使用肾上腺嗜铬细胞和PC 12细胞确定胞吐途径中的边缘功能。将使用生物化学和电生理技术。我们已经确定了两个域，独立增强分泌时，在嗜铬细胞，锌指结构域和C2b结构域的表达。我们已经确定了两个合理的锌指结构域的配体。这些蛋白质与锌指结构域的相互作用和分泌途径上的相互作用的后果将被调查。我们还将研究C2结构域的配体的相互作用的功能意义，这已经被其他人确定，并寻找其他配体。一个相关的问题，内源性Rim 2在嗜铬细胞和PC 12细胞中的功能也将被研究。用海马神经元培养物进行的实验将研究Rim/Rab 3a相互作用和Rim 1 PDZ结构域在转染的Rim 1在神经末梢中的定位中的作用。

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.