Calcium Regulation of Secretion in Neuroendocrine Cells

神经内分泌细胞分泌的钙调节

• 批准号: 8639526
• 负责人: THOMAS F. J. MARTIN
• 金额: $ 32.01万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-10 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8639526
• 关键词: Address; Artificial Membranes; Binding; Binding Proteins; Biochemical; Biochemical Genetics; Biological; Calcium; Cell membrane; Cells; Competence; Complex; Dense Core Vesicle; Disease; Docking; Endocrine; Endocrine system; Event; Exocytosis; Family; Fluorescence Resonance Energy Transfer; Genetic; Homologous Protein; Image; In Vitro; Inflammation Mediators; Knowledge; Lead; Link; Location; Mediating; Membrane; Membrane Fusion; Molecular; Mutagenesis; Mutation; Nervous system structure; Neuroendocrine Cell; Neurons; Neuropeptides; Neurotransmitters; PC12 Cells; Pathway interactions; Physiological; Play; Positioning Attribute; Process; Protein Family; Proteins; Regulation; Research; Role; S-nitro-N-acetylpenicillamine; SNAP receptor; Signaling Molecule; Site; Staging; Synaptic Vesicles; Synaptic plasticity; Total Internal Reflection Fluorescent; VAMP-2; Vesicle; Work; base; insight; mast cell; member; monomer; mutant; peptide hormone; prevent; public health relevance; reconstitution; relating to nervous system; synaptotagmin; syntaxin; syntaxin 1; target SNARE proteins; therapy development; vesicle-associated membrane protein

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.

描述（由申请人提供）：神经递质、神经肽和肽激素的分泌通过Ca 2+依赖性囊泡胞吐作用发生。介导囊泡与质膜融合的机制被充分表征，并且由囊泡SNARE VAMP-2/小突触泡蛋白与质膜SNARE突触融合蛋白-1和SNAP-25的复合物组成。SNARE复合物被Ca 2+结合的突触结合蛋白作用以驱动膜融合。虽然Ca 2+触发囊泡融合的这些最后步骤已被广泛研究，但在理解融合前的关键事件方面仍存在重要差距。这些事件被称为引发，它们赋予停靠的囊泡用于Ca 2+触发融合的能力。引发被认为涉及反式SNARE复合物的逐步组装，但所利用的精确途径和调节它的因子尚未被表征。遗传和生物化学研究表明，CAPS/Munc 13蛋白家族的成员在引发中起作用。我们的研究将从分子水平阐明CAPS在启动致密核心囊泡（DCV）胞吐中的作用机制。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在体外促进反式SNARE复合物的形成，并与3种SNARE蛋白中的每一种相互作用。此外，CAPS经历四聚体形成，这表明催化SNARE复合物组装的机制。CAPS存在于质膜上和DCV上的中心位置，用于控制DCV胞吐。我们提出了生物化学，细胞生物学和生物物理学研究介导的启动CAPS功能的分子事件。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，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的反式SNARE复合物组装;（3）确定CAPS寡聚化和DCV结合是否对CAPS启动功能起关键作用。工作的重点包括成像CAPS和SNARE在网站上的胞吐，重建启动人工膜上的CAPS，和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一个分子描述的启动和填补了一个重要的空白，我们的理解调节囊泡胞吐的途径。