Regulation of tomosyn-SNARE complex assembly in neurosecretion

神经分泌中 tomosyn-SNARE 复合体组装的调节

• 批准号: 7278182
• 负责人: EDWARD L STUENKEL
• 金额: $ 31.06万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-20 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7278182
• 关键词: Adrenal Glands; Agonist; Binding; Biochemical; Bos taurus; C-terminal; Cattle; Cell membrane; Cell model; Cells; Characteristics; Chromaffin Cells; Complex; Condition; Cyclic AMP-Dependent Protein Kinases; Cytoplasmic Granules; Dependence; Development; Electrophysiology (science); Equilibrium; Event; Exocytosis; Exocytosis Inhibition; Fluorescence; Goals; Guanosine Triphosphate Phosphohydrolases; Hippocampus (Brain); Kinetics; Mediating; Membrane; Membrane Microdomains; Molecular; N-terminal; Nervous System Physiology; Neurites; Neurologic; Neurons; Neurosecretion; Neurotransmitters; Optics; PC12 Cells; Pathway interactions; Phosphorylation; Photobleaching; Property; Protein Overexpression; Proteins; Regulation; Reporting; Research; Research Personnel; Rho-associated kinase; SNAP receptor; Secretory Vesicles; Signal Pathway; Signal Transduction Pathway; Site; Small Interfering RNA; Testing; Therapeutic; Thinking; Time; dimer; effusion; neurotransmitter release; programs; research study; rho; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; vesicle-associated membrane protein

DESCRIPTION (provided by applicant): The assembly of SNARE proteins into a ternary core complex is essential for neurotransmitter release. Precise regulation of SNARE complex assembly ultimately determines the site and dynamics of the exocytotic event. Our objective is to understand the mechanisms that regulate temporal and spatial assembly of these SNARE complexes. Tomosyn is a protein that is critical in setting the level effusion- competent SNARE complexes. Its regulatory action has been proposed to be primarily mediated by its interaction with the Q-SNARE syntaxinl A, which results in the formation of non-fusogenic SNARE complexes. The goal of the research proposed is to provide an understanding of the molecular mechanisms and signal transduction pathways governing the assembly/disassembly of tomosyn-SNARE complexes in the regulated release of neurotransmitter. Our general hypothesis is that tomosyn-SNARE complex formation is promoted by Rho-GTPase signaling pathways and antagonized by protein kinase A-dependent pathways, with the balance of activation of these pathways fine-tuning the level of fusion-competent SNARE complexes. Aim 1 will test the hypothesis that the formation of tomosyn-SNARE complexes is under dynamic control by secretory agonists to regulate secretion. In addition, we will identify N-terminal tomosyn domains important for this regulation, and quantity effects of endogenous tomosyn on granule priming and exocytosis. Aim 2 will test the hypothesis that the level of tomosyn-SNARE complexes and their functional effects on the exocytotic pathway are finely regulated by a balance between the activation state of Rho, to promote tomosyn-SNARE complex assembly, and PKA, to antagonize tomosyn-SNARE complex assembly. Aim 3 will define the spatial properties of tomosyn-SNARE complexes on the plasma membrane and determine if RhoA and PKA mediate spatially delimited effects on the assembly and disassembly of these complexes. Experiments will be performed using a combination of biochemical, optical and electrophysiological approaches on adrenal chromaffin cells, a highly characterized cell model for neurotransmitter release. Understanding the regulation of neurotransmitter release is essential to understanding the function of the nervous system and fundamental to development of therapeutic treatments in the many psychiatric and neurological conditions typified by an imbalance of particular neurotransmitters.

描述（由申请人提供）：将 SNARE 蛋白组装成三元核心复合物对于神经递质释放至关重要。 SNARE 复合体组装的精确调节最终决定了胞吐事件的位点和动态。我们的目标是了解调节这些 SNARE 复合体的时间和空间组装的机制。 Tomosyn 是一种对于设置具有渗出能力的 SNARE 复合物水平至关重要的蛋白质。其调节作用被认为主要是通过其与 Q-SNARE 突触蛋白 A 的相互作用介导的，从而导致非融合 SNARE 复合物的形成。本研究的目的是了解在神经递质调节释放中控制 tomosyn-SNARE 复合物组装/分解的分子机制和信号转导途径。我们的一般假设是，Rho-GTPase 信号通路促进 Tomosyn-SNARE 复合物的形成，并受到蛋白激酶 A 依赖性通路的拮抗，这些通路的激活平衡可微调具有融合能力的 SNARE 复合物的水平。目标 1 将检验以下假设：断层合成-SNARE 复合物的形成受到分泌激动剂动态控制以调节分泌。此外，我们将确定对于这种调节很重要的 N 端断层合成结构域，以及内源性断层合成对颗粒引发和胞吐作用的数量影响。目标 2 将检验这样的假设，即 Tomosyn-SNARE 复合物的水平及其对胞吐途径的功能影响通过 Rho 激活状态（促进 tomosyn-SNARE 复合物组装）和 PKA（拮抗 tomosyn-SNARE 复合物组装）之间的平衡进行精细调节。目标 3 将定义质膜上 tomosyn-SNARE 复合物的空间特性，并确定 RhoA 和 PKA 是否介导这些复合物组装和解体的空间界定效应。实验将结合生化、光学和电生理学方法在肾上腺嗜铬细胞上进行，肾上腺嗜铬细胞是一种高度特征化的神经递质释放细胞模型。了解神经递质释放的调节对于了解神经系统的功能至关重要，也是开发以特定神经递质失衡为代表的许多精神和神经疾病的治疗方法的基础。