Rim and Exocytosis in Chromaffin Cells

嗜铬细胞的边缘和胞吐作用

• 批准号: 6755111
• 负责人: RONALD W HOLZ
• 金额: $ 27.27万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 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/6755111
• 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

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讯和突触传递的核心，也是内分泌、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。本提案将研究其在胞吐作用中的功能。 Rim1 是一种多结构域 Rab3a 结合蛋白，位于神经末梢的活性区域。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。 Rim1 及其密切同源的家族成员 Rim2 强烈增强嗜铬细胞分泌的生化测定中的胞吐作用。指导这一提议的假设是，多模块蛋白 Rim1 和 Rim2 是分子支架，整合了对胞吐作用至关重要的蛋白质的功能。一个基本假设是，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，而且还将阐明协调这一复杂的多步骤过程的重要途径。胞吐途径中的 Rim 功能将主要使用肾上腺嗜铬细胞和 PC12 细胞来确定。将使用生物化学和电生理学技术。我们已经鉴定了在嗜铬细胞中表达时独立增强分泌的两个结构域：Zn 指结构域和 C2b 结构域。我们已经确定了锌指结构域的两个可能的配体。将研究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。我们还将研究其他人已鉴定的 C2 结构域配体相互作用的功能意义，并寻找其他配体。一个相关的问题，内源性 Rim2 在嗜铬细胞和 PC12 细胞中的功能也将得到研究。海马神经元培养实验将研究 Rim/Rab3a 相互作用以及 Rim1 PDZ 结构域在转染的 Rim1 在神经末梢定位中的作用。