TRANSMEMEBRANE SIGNALING VIA RECEPTORS AND G PROTEINS

GTP依赖的调节蛋白(G蛋白)调节许多 细胞内过程，包括许多受 细胞外刺激(如激素、光线、气味等)。 例如，异三聚体G蛋白调节荷尔蒙 效应分子的调节，如腺酰环化酶或 磷脂酶C(PLC)改变第二信使的产生， CAMP或肌醇1，4，5-三磷酸和二酰甘油， 分别进行了分析。单体G蛋白，如Ras、Rho或Rab 家庭调节生长因素的影响或更直接地在 调节细胞形状或细胞内的囊泡运输， 分别进行了分析。这项研究的长期目标过去和现在都是 阐明了调控的途径和机制 G蛋白。 在异源三聚体G蛋白中，普遍表达的 由G12和G13组成的亚家族脱颖而出 特色化的。在这一群体中的进展导致了 G13的纯化及其在细胞周期调控中的作用 钙离子通道的调节。然而，紧随其后的下游 G13的效应器尚不清楚。此应用程序的一个目标是 进一步表征纯化的G13及其产物的性质 与受体的相互作用。对G13遗嘱功能的考察 包括Pbenotype特征互补组合 细胞过度表达蛋白质和生化方法 分离和鉴定受G13调控的效应蛋白。 许多刺激PLC活性的激素也会增加PLC的活性 磷脂酶D(PLD)的活性，从而产生 磷脂酸(PA)，一种可能的第二信使和前体 二酰甘油和溶血磷脂酸(LPA)。最新进展 证明了PLD的活性可以由一个小的 单体G蛋白，ARF。此应用程序建议检查 PLD监管的几个方面。目标包括提纯， ARF敏感的PLD(ARF-PLD)酶的克隆和表达， 并对粗酶和纯化酶进行了鉴定。 对结构、催化活性和ARF等的调节 潜在的因素。进一步的追求包括尝试 描述荷尔蒙调节的PLD活性，可能包括 直接调控的新型酶的鉴定 异三聚体G蛋白或其决定机制 受体刺激ARF/PLD。有越来越多的证据表明， ARF在细胞内蛋白运输中的作用。因此， 还建议进行实验以确定其潜在的作用 ARF-PLD及其机制中膜脂的电位变化 用于水泡流量。 这些实验的进展将有助于进一步 G蛋白机制的描述以及我们对G蛋白的理解 荷尔蒙的调节。ARF对PLD调节的研究可能 进一步阐明细胞内的基本机制 蛋白质的运输和分泌。

GTP-dependent regulatory proteins (G proteins) modulate numerous intracellular processes including many that are regulated by extracellular stimuli (such as hormones, light, odorants, etc.). For example, the heterotrimeric G proteins mediate hormonal regulation of effector molecules such as adenylyl cyclase or phospholipase C (PLC) to alter production of the second messengers, cAMP or inositol 1,4,5-trisphosphate and diacylglycerol, respectively. Monomeric G proteins such as the Ras, Rho, or Rab families mediate effects of growth factors or act more directly in regulating cell shape or intracellular vesicle traffic, respectively. The long term goal of this research has been and is the elucidation of pathways and mechanisms for regulation through the G proteins. Among the heterotrimeric G proteins, the ubiquitously expressed subfamily consisting of G12 and G13 stands out as the least characterized. Progress in this group has resulted in the purification of G13 and identification of its role in the regulation of Ca2+ channels. Nevertheless, the immediate downstream effector of G13 is unknown. One goal of this application is to further characterize the properties of purified G13 and its interaction with receptors. Examination of the function of G13 will include a complementary combination of pbenotypic characterization of cells overexpressing the protein and biochemical approaches to isolate and characterize effector proteins regulated by G13. Many hormones which stimulate the activity of PLC also increase the activity of phospholipase D (PLD) and thus, the production of phosphatidic acid (PA), a putative second messenger and precursor to diacylglycerol and lysophosphatidic acid (LPA). Recent progress demonstrated that PLD activity could be regulated by a small monomeric G protein, ARF. This application proposes to examine several aspects of PLD regulation. Aims include purification, cloning, and expression of the ARF-sensitive PLD (ARF-PLD) enzyme, and characterization of the crude and purified enzyme with respect to structure, catalytic activity, and regulation by ARF and other potential factors. Further pursuits include attempts to characterize hormone regulated PLD activities which could include identification of novel enzymes directly regulated by heterotrimeric G proteins or determining mechanism by which receptors stimulate ARF/PLD. Evidence has mounted for a prominent role of ARF in intracellular protein trafficking. Therefore, experiments are also proposed to determine the potential role of ARF-PLD and potential changes in membrane lipids in the mechanisms for vesicle traffic. Progress from these experiments will contribute to further delineation of G-protein mechanisms and thus our understanding of regulation by hormones. The study of PLD regulation by ARF may further elucidate fundamental mechanisms for intracellular transport and secretion of proteins.