TRANSMEMEBRANE SIGNALING VIA RECEPTORS AND G PROTEINS

GTP依赖性调节蛋白（G蛋白）调节许多 细胞内过程包括许多由 细胞外刺激（如激素、光、气味等）。 例如，异源三聚体G蛋白介导激素分泌， 调节效应分子如腺苷酸环化酶或 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基甘油， 分别单体G蛋白，如Ras、Rho或Rab 家庭介导生长因子的作用，或更直接地作用于 调节细胞形状或胞内囊泡运输， 分别这项研究的长期目标一直是， 阐明通过以下途径进行调节的途径和机制 G蛋白。 在异源三聚体G蛋白中，普遍表达的 由G12和G13组成的亚家族是最不突出的 表征了这一组的进展导致了 G_（13）的纯化及其在细胞凋亡中的作用 调节Ca 2+通道。尽管如此， G13的效应子未知。此应用程序的一个目标是 进一步表征纯化的G13及其 与受体的相互作用。对G13职能的审查将 包括表型特征互补组合 细胞过度表达蛋白质和生化方法， 分离和表征由G13调节效应蛋白。 许多刺激PLC活性的激素也增加了PLC的活性。 磷脂酶D（PLD）的活性，从而产生 磷脂酸（PA），推定的第二信使和前体 二酰基甘油和溶血磷脂酸（LPA）。最新进展 表明PLD活性可以通过一个小的 单体G蛋白。本申请旨在审查 PLD法规的几个方面。目的包括纯化， 克隆和表达ARF-敏感PLD（ARF-PLD）酶， 以及粗酶和纯化酶的表征， 结构，催化活性，以及ARF和其他 潜在因素。进一步的努力包括： 表征激素调节的PLD活性，其可包括 鉴定直接受 异源三聚体G蛋白或决定机制， 受体刺激ARF/PLD。有证据表明 ARF在细胞内蛋白运输中的作用。因此，我们认为， 还提出了实验来确定潜在的作用， ARF-PLD与潜在膜脂变化的机制 用于囊泡运输。 这些实验的进展将有助于进一步 描述G蛋白的机制，从而我们对 受激素调节。ARF对PLD调控的研究可能 进一步阐明细胞内 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.