RAS LIKE GTP BINDING PROTEINS--REGULATION AND FUNCTION

描述：这是一个修改后的应用程序，用于研究 Rab3 GTP酶在神经内分泌细胞胞吐调节中的作用 Rab3A在Rab GTPase成员中是不寻常的，因为它是 仅在神经内分泌细胞中表达，在那里它是定位的 主要分布在神经元的突触颗粒和致密的分泌颗粒上 在PC12和肾上腺嗜铬细胞中。Rab3A基因的亚细胞定位 结合另外两项观察表明，这种蛋白质在 胞吐。第一个观察是野生型基因的过度表达 Rab3A，或被激活的Q81突变体，有效地抑制了 激动剂刺激的分泌。第二个观察结果是删除了一个 Rab3A可能的效应者RabPhilin的结构域也抑制了 分泌物。马卡拉博士建议Rab3A靶向RabPhilin到囊泡 膜，在这一点上胞浆钙可以促进结合 拉普林以细胞骨架为前传，与囊泡对接。另一个 与Rab3A的作用相关的蛋白质是Rabin3，它参与了 Rab3-鸟嘌呤核苷酸交换活性与Rab3的关系。 在此背景下，该提案的具体目标是：1) 确定Rabin3是否与Rab3特异的核苷酸交换相互作用 因素，以及膜上的这种因素是否优先作用于 Rab3：GDI复合体；2)确定Rab3A是否靶向RabPhilin 分泌泡泡膜，或反之亦然；3)鉴定 RabPhilin结构域，特别是C2结构域的结构域，并搜索 RabPhilin结合蛋白；4)研究细胞功能 Rab3A：RabPhilin复合体，即突变体对分布的影响 分泌囊泡及其与Rab3A结合对其相互作用的影响 含有β-内收蛋白和其他蛋白质的RabPhilin；以及5)搜索 表达Rab3异构体的组织中的交替下游效应物 而不是Rab3A。

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.