Calcium Regulation of Secretion in Neuroendocrine Cells

描述（由申请人提供）：神经递质、神经肽和肽激素的分泌通过Ca2+依赖性囊泡胞吐发生。介导囊泡与质膜融合的机制是很有特点的，它由囊泡SNARE VAMP-2/synaptobrevin与质膜SNARE syntaxin-1和SNAP-25的复合物组成。SNARE复合体由Ca2+结合的synaptotagmin作用来驱动膜融合。虽然Ca2+触发囊泡融合的这些最后步骤已被广泛研究，但在理解融合之前的关键事件方面仍然存在重要差距。这些事件被称为引物，它们赋予对接囊泡的能力，以进行Ca2+触发的融合。启动被认为涉及跨SNARE复合物的逐步组装，但所利用的确切途径和调节它的因素尚未被表征。遗传和生化研究表明，CAPS/Munc13蛋白家族成员参与了启动。我们提出的研究将获得CAPS功能在启动密核囊泡（DCV）胞吐作用中的分子机制。这项工作是基于上一个项目期间的重大进展，该项目发现CAPS在体外促进反SNARE复合物的形成，并与3种SNARE蛋白相互作用。此外，CAPS可以形成四聚体，这表明了催化SNARE复合物组装的机制。CAPS存在于质膜和DCV的中心位置，用于控制DCV胞吐。我们建议从生物化学、细胞生物学和生物物理学的角度研究介导CAPS启动的分子事件。我们的具体目标将是：(1)确定CAPS的启动活性是否通过其与VAMP-2、syntaxin-1和SNAP-25的相互作用介导；(2)确定CAPS是否促进神经内分泌细胞中trans SNARE复合物的组装；(3)确定CAPS寡聚化和DCV结合是否对CAPS启动功能起关键作用。工作的重点包括胞外作用部位的CAPS和SNAREs成像，人工膜上CAPS的启动重构，CAPS四聚体及其snare结合域的结构研究。这项工作的完成将提供启动的分子描述，并填补我们对调控囊泡胞吐途径的理解的重要空白。

DESCRIPTION (provided by applicant): The secretion of neurotransmitters, neuropeptides and peptide hormones occurs by Ca2+dependent vesicle exocytosis. The machinery that mediates vesicle fusion with the plasma membrane is well-characterized and consists of complexes of the vesicle SNARE VAMP-2/synaptobrevin with the plasma membrane SNAREs syntaxin-1 and SNAP-25. SNARE complexes are acted upon by Ca2+bound synaptotagmin to drive membrane fusion. While these final steps in Ca2+triggered vesicle fusion have been extensively studied, there remain important gaps in understanding key events that precede fusion. These events are termed priming and they confer competence to docked vesicles for Ca2+triggered fusion. Priming is thought to involve the progressive assembly of trans SNARE complexes but the precise pathway utilized and the factors that regulate it have not been characterized. Genetic and biochemical studies indicate that members of the CAPS/Munc13 family of proteins operate in priming. Our proposed research will obtain a molecular description of the mechanism of CAPS function in priming dense-core vesicle (DCV) exocytosis. This work is based on major advances during the previous project period, which discovered that CAPS promotes trans SNARE complex formation in vitro and interacts with each of the 3 SNARE proteins. Moreover, CAPS undergoes tetramer formation, which suggests a mechanism for catalyzing SNARE complex assembly. CAPS is present on the plasma membrane and on DCVs in a central location for controlling DCV exocytosis. We propose biochemical, cell biological and biophysical studies on the molecular events that mediate CAPS function in priming. Our specific aims will be to: (1) determine whether the priming activity of CAPS is mediated through its interactions with VAMP-2, syntaxin-1 and SNAP-25; (2) determine whether CAPS promotes trans SNARE complex assembly in neuroendocrine cells; and (3) determine whether CAPS oligomerization and DCV binding play critical roles for CAPS function in priming. Highlights of the work include imaging of CAPS and SNAREs at sites of exocytosis, reconstituting priming by CAPS on artificial membranes, and structural studies of CAPS tetramers and its SNARE-binding domain. Completion of this work will provide a molecular description of priming and fill an important gap in our understanding of the pathway for regulated vesicle exocytosis. PUBLIC HEALTH RELEVANCE: Neurotransmitters, peptide hormones, and inflammatory mediators are secreted from neural, endocrine and mast cells by the regulated fusion of vesicles with the plasma membrane. We will study key proteins that are needed for this process. Because a variety of diseases involve excessive or inadequate secretion, this work will contribute basic knowledge about these conditions and set the stage for developing therapies.