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.
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