RAS LIKE GTP BINDING PROTEINS--REGULATION AND FUNCTION

RAS 样 GTP 结合蛋白——调控和功能

• 批准号: 2712655
• 负责人: IAN G MACARA
• 金额: $ 24.34万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-15 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2712655
• 关键词: PC12 cells; exocytosis; genetic regulatory element; guanine nucleotide binding protein; guanine nucleotide exchange factors; guanosinetriphosphatases; immunocytochemistry; immunoelectron microscopy; molecular cloning; neuroendocrine system; protein isoforms; protein structure function; protein transport; transfection

DESCRIPTION: This is a revised application to investigate the function of the Rab3 GTPase in the regulation of exocytosis from neuroendocrine cells. Rab3A is unusual among members of the Rab GTPases in the sense that it is expressed exclusively in neuroendocrine cells, where it is localized predominantly on synaptic granules in neurons and dense secretory granules in PC12 and adrenal chromaffin cells. The subcellular location of Rab3A together with two other observations suggest a role for the protein in exocytosis. The first observation is that overexpression of wild-type Rab3A, or the activated Q81 mutant, potently inhibits the initial rate of agonist-stimulated secretion. The second observation is that removal of a domain from a putative effector of Rab3A, Rabphilin, also inhibits secretion. Dr. Macara proposes that Rab3A targets Rabphilin to vesicle membrane, at which point cytosolic calcium can promote an association of Rabphilin with the cytoskeleton as a prequel to vesicle docking. Another protein relevant to the actions of Rab3A is Rabin3, which is implicated in the association of Rab3-guanine nucleotide exchange activity with Rab3. With that as the background, the specific aims of the proposal are: 1) to determine whether Rabin3 interacts with a Rab3-specific nucleotide exchange factor, and whether such a factor on membranes preferentially acts on a Rab3:GDI complex; 2) to determine whether Rab3A targets Rabphilin to secretory vesicle membrane, or vice versa; 3) to identify the functions of the Rabphilin domains, particularly that of the C2 domain, and to search for Rabphilin-binding proteins; 4) to investigate the cellular function of the Rab3A:Rabphilin complex, that is, effects of mutants on the distribution of secretory vesicles and the effects of Rab3A binding on the interaction of Rabphilin with beta-adducin and other proteins; and 5) to search for alternate downstream effectors in tissues that express Rab3 isoforms other than Rab3A.

描述：这是一个修订后的应用程序，用于研究 Rab 3 GT3在调节神经内分泌细胞胞吐作用中的作用。 Rab 3A在Rab GTP酶的成员中是不寻常的，因为它是 仅在神经内分泌细胞中表达， 主要分布在神经元内的突触颗粒和致密分泌颗粒上 在PC 12和肾上腺嗜铬细胞中。 Rab 3A的亚细胞定位 与另外两个观察结果一起表明，蛋白质在 胞吐作用 第一个观察结果是野生型的过度表达 Rab 3A，或激活的Q81突变体，有效地抑制初始速率， 激动剂刺激的分泌。 第二个观察结果是， 来自Rab 3A的假定效应子Rabphilin的结构域也抑制 分泌物 Macara博士提出Rab 3A将Rabphilin靶向于囊泡 膜，在这一点上，细胞溶质钙可以促进协会 Rabphilin与细胞骨架作为囊泡对接的前传。 另一 与Rab 3A的作用相关的蛋白是Rabin 3，其涉及 Rab 3-鸟嘌呤核苷酸交换活性与Rab 3. 在此背景下，建议的具体目的是：1） 确定Rabin 3是否与Rab 3特异性核苷酸交换相互作用 因子，以及膜上的这种因子是否优先作用于 Rab 3：GDI复合物; 2）确定Rab 3A是否靶向Rabphilin， 分泌囊泡膜，或反之亦然; 3）确定的功能， Rabphilin结构域，特别是C2结构域，并寻找 Rabphilin结合蛋白; 4）研究Rabphilin结合蛋白的细胞功能。 Rab 3A：Rabphilin复合物，即突变体对Rab 3A分布的影响。 分泌囊泡和Rab 3A结合的相互作用的影响， Rabphilin与β-内收蛋白和其他蛋白质;和5）寻找 表达Rab 3同种型的组织中的替代下游效应物 比Rab 3A。