IMMUNOLOCALIZATION OF G PROTEINS IN MAMMALIAN CELLS

异三聚体GTP结合蛋白(G蛋白)起着至关重要的作用 通过质膜传递受体产生的信号。 最具特性的信号转导途径之一 激素敏感的腺酰环化酶系统。效应酶是在 G和G两种G蛋白的双重刺激和抑制调控 然而，现在已经知道，这些G蛋白中的每一个都在 除了腺酰环化酶的调节外，在G的情况下，还有 是三种密切相关的基因产物，可以在一个 单元格的类型。G的每一种不同形式的功能不是 为人所知。对信号转导机制的理解是基础 对医学的许多方面，包括激素和药物作用。更改后的 在心力衰竭等疾病中观察到G蛋白活性， 高血压和癌症。了解特定G的位置 蛋白质可能提供对这种临床相关性的洞察 观察。关于G的亚细胞分布的最多信息 蛋白质来自相对原始的细胞尝试 分馏，几乎没有关于它们的超微结构信息 重要的话题。G蛋白活性，尽管明显存在于 质膜，已被报道与细胞内有关 隔室，包括高尔基体，线粒体，内体， 细胞核，并在激活后形成细胞质。1)第一个具体 目的是利用超薄冰冻切片的免疫电子显微镜 定量鉴定G的亚细胞结构 蛋白质亚单位在细胞中是相关的。我们将尝试 确定质膜上的特殊结构位置和 由G和每种形式的G填充的细胞膜。 待检验的假设是个体的差异分布 G蛋白寡聚体有助于其参与的特异性 在信号转导途径和G蛋白知识中 本地化将阐明它们的功能角色。2)第二个 具体目标包括审查已定义的结构的作用 膜定位中α亚基的特点。分布情况 内源性G蛋白的数量将用定量的 全细胞和血浆的形态和生化技术 暴露在已知可改变G蛋白功能的试剂中的细胞膜。基于 根据这些实验的结果，突变的G蛋白α亚基 处于活动和非活动状态。3)第三个具体目标是 确定受体的局部化与 它们的附属G蛋白。将检查受体和G蛋白 用于区分分布到基侧或根尖质膜 上皮细胞。此外，G在脱敏过程中的命运 可能为了解G蛋白的其他功能以及 解释信号转导中G蛋白特异性的机制。

Heterotrimeric GTP binding proteins (G proteins) serve an essential role in transducing receptor-generated signals across the plasma membrane. One of the best characterized signal transduction pathways in the hormone-sensitive adenylyl cyclase system. The effector enzyme is under dual stimulatory and inhibitory control by two G proteins termed G and G. However, it is now known that each of these G proteins has roles in addition to regulation of adenylyl cyclase and, in the case of G there are three closely related gene products that can be expressed in a single type of cell. The function of each of the different forms of G is not known. An understanding of signal transduction mechanisms is fundamental to many aspect of medicine including hormone and drug action. Altered G protein activity has been observed in diseases such as heart failure, hypertension, and cancer. Knowledge of the location of specific G proteins may provide insight into the clinical relevance of such observation. Most information about the subcellular distribution of G proteins has come from relatively crude attempts at cellular fractionation, and there is little ultrastructural information on their important subject. G protein activity, although clearly present in the plasma membrane, has been reported to be associated with intracellular compartment, including the Golgiapparatus,mitochondria, endosomes, nucleus, and following activation, the cytoplasm. 1) The first specific aim is to utilize immunoelectron microscopy of ultrathin frozen sections to identify quantitatively the subcellular structures with which G protein subunits are associated in cells. Attempts will be made to identify specialized structural sites on the plasma membrane and intracellular membranes that are populated by G and each form of G. The hypothesis to be tested is hat differential distribution of individual G protein oligomers contributes to the specificity of their participation in signal transduction pathways and that knowledge of G protein localization will shed light on their functional roles. 2) The second specific aim involves examination of the role of defined structural features of alpha subunits in membrane localization. The distribution of endogenous G proteins will be examined using quantitative morphological and biochemical techniques on whole cells and plasma membranes exposed to agents known to alter G protein function. Based in part on the results of these experiments, mutant G protein alpha subunits in the active and inactive states. 3) The third specific aim is to determine the relationship between the localization of receptors and their affiliated G proteins. Receptors and G proteins will be examined for differential distribution to basolateral or apical plasma membranes of epithelial cells. In addition, the fate of G during desensitization may provide insight into additional functions of G proteins as well as mechanisms to explain G protein specificity in signal transduction.