Rim and Exocytosis in Chromaffin Cells

描述（由申请人提供）：激素和神经递质的胞外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受调节的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外分泌的重要调节因子。本文将探讨其在胞吐中的作用。Rim1是一种多结构域rab3a结合蛋白，位于神经末梢的活性区。基因敲除实验揭示秀丽隐杆线虫和小鼠突触传递的缺陷。在嗜铬细胞分泌的生化测定中，Rim1及其近亲家族成员Rim2强烈地促进胞吐作用。指导这一建议的假设是，多模块蛋白Rim1和Rim2是分子支架，整合了对胞吐至关重要的蛋白质的功能。一个潜在的假设是，识别与Rim中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，还可以阐明这一复杂的多步骤过程协调的基本途径。外胞途径中的Rim功能将主要通过肾上腺染色质和PC12细胞来确定。将使用生化和电生理技术。我们已经确定了两个结构域，Zn指结构域和C2b结构域，当在染色质细胞中表达时，它们独立地促进分泌。我们已经确定了锌指结构域的两种合理的配体。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结构域的配体相互作用的功能意义，这些配体已经被其他人发现，并寻找其他配体。一个相关的问题，内源性Rim2在染色质和PC12细胞中的功能也将被研究。海马神经元培养实验将探讨Rim/Rab3a相互作用和Rim1 PDZ结构域在转染后的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.