Calcium Regulation of Secretion in Neuroendocrine Cells

描述(申请人提供)：神经递质、神经肽和多肽激素的分泌是通过依赖于钙离子的囊泡胞吐来实现的。介导囊泡与质膜融合的机制是由囊泡陷阱VAMP-2/Synaptobrevin与质膜陷阱Synaxin-1和SNAP-25组成的复合体。SNARE复合体被钙离子结合的突触凝集素作用于膜融合。虽然钙离子触发的囊泡融合的这些最后步骤已经被广泛研究，但在理解融合之前的关键事件方面仍然存在重要的差距。这些事件被称为启动，它们赋予停靠的小泡进行钙离子触发的融合的能力。启动被认为涉及反式SNARE复合体的渐进组装，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家族的成员在启动过程中起作用。我们提出的研究将获得CAPS在启动致密核小泡(DCV)胞吐中的作用机制的分子描述。这项工作是基于前一个项目期间的主要进展，发现CAPS在体外促进反式SNARE复合体的形成，并与3种SNARE蛋白中的每一种相互作用。此外，CAPS还经历了四聚体的形成，这表明了一种催化SNARE络合物组装的机制。CAPS存在于质膜和DCV上，处于控制DCV胞吐的中心位置。我们建议对介导CAPS启动功能的分子事件进行生化、细胞生物学和生物物理研究。我们的具体目标是：(1)确定CAPS的启动活性是否通过其与VAMP-2、Synaxin-1和SNAP-25的相互作用来介导；(2)确定CAPS是否促进神经内分泌细胞中反式SNARE复合体的组装；以及(3)确定CAPS寡聚和DCV结合是否在启动CAPS功能中起关键作用。这项工作的亮点包括对胞吐部位的帽和陷阱的成像，通过人造膜上的帽重新启动，以及帽四聚体及其陷阱结合结构域的结构研究。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一个重要空白。

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.