Calcium Regulation of Secretion in Neuroendocrine Cells

神经内分泌细胞分泌的钙调节

• 批准号: 8443850
• 负责人: THOMAS F. J. MARTIN
• 金额: $ 30.89万
• 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/8443850
• 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发生在Ca2+依赖性囊泡胞吐作用中。与质膜介导囊泡融合的机械是良好的特征，并由囊泡SNARE VAMP-2/Synaptobrevin与质膜snares stytaxin-1和Snap-25组成。 SNARE复合物由Ca2+结合突触抗体作用，以驱动膜融合。尽管已经对Ca2+触发囊泡融合的这些最后一步进行了广泛的研究，但在理解融合之前的关键事件中仍然存在重要差距。这些事件被称为启动，它们赋予能力与Ca2+触发的融合的囊泡对接。启动被认为涉及反式SNARE复合物的渐进式组装，但是所使用的精确途径及其调节的因素尚未被表征。遗传学和生化研究表明，蛋白质蛋白质蛋白质的成员在启动中起作用。我们提出的研究将获得对蛋白质机理在启动致密囊泡（DCV）胞吐作用中功能的分子描述。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在体外促进了反式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经历四聚体的形成，这提出了一种催化军鼓组装的机制。盖帽存在于质膜上，并且在中心位置，用于控制DCV胞吐作用。我们提出了介导盖在启动中起作用的分子事件的生化，细胞生物学和生物物理研究。我们的具体目的是：（1）确定CAP的启动活性是否是通过与VAMP-2，Syntaxin-1和SNAP-25的相互作用来介导的； （2）确定CAP是否促进了神经内分泌细胞中的反式SNARE复合物组装； （3）确定CAPS寡聚和DCV结合在启动中的cap函数是否起关键作用。这项工作的亮点包括在胞吞作用部位的帽子和网罗成像，帽子在人造膜上重新建立启动，以及对四聚体的帽子及其网状结合域的结构研究。这项工作的完成将提供启动的分子描述，并填补我们对调节囊泡胞吐作用途径的重要空白。