LOCALIZATION OF G PROTEIN ALPHA SUBUNITS IN EPITHELIUM

鸟嘌呤核苷酸结合蛋白（G蛋白）是一种普遍存在的蛋白质 它能抑制来自离子通道、生长因子、激素和 神经递质转化为各种细胞内效应物。 体外 用纯化的受体、G蛋白和效应物进行的重建研究 表明几种不同的G蛋白可以与受体相互作用， 和效应器。 其他机制在体内发挥作用，以限制 各种可能的互动。 可以实现这一点的一种方式是 将G蛋白定位于特定的质膜结构域。 许多 细胞包括上皮细胞分离质膜蛋白 （包括一些G蛋白）到不同的表面。 这项建议的广泛的长期目标是确定 上皮细胞定位G蛋白亚单位的机制 与特定的质膜结构域结合， 特异性膜所必需的α亚单位的要求 面向. 我们的目标是为这项研究建立一个细胞培养模型 特定的膜定位。 α 12定位于基底外侧， MDCK和LLC-PK 1细胞都将被标记上来自 不相关的蛋白质，以区分转染的内源性亚基。 标记的alpha 12亚基的体外表征将允许α 亚基性质，包括与β-γ的相互作用， GTP和肉豆蔻酰化有待研究。 表位标记的α i2将是 其特征在于确定免疫沉淀的条件， 抗表位抗体。 标记的α i2将被定位在细胞中， 免疫荧光和Western印迹。 Alpha的结构特征 将使用α 12中的突变来定义正确排序所必需的 基于我之前对阿尔法欧米克隆的体外研究 氨基和 标记α 12和嵌合α 12中的羧基末端突变 蛋白质将被用来定义这些区域。 的定时和 将使用脉冲追踪标记定义分选途径 实验和选择性膜纯化。 免疫沉淀 使用纯化的囊泡的研究将开始定义其他细胞 参与排序过程的元素。 G蛋白对许多细胞过程至关重要，G蛋白的变化 蛋白质与包括霍乱在内的几种疾病有关， 奥尔布赖特遗传性骨营养不良 激活alpha/ s突变 和α/I在几种人类内分泌肿瘤中有描述。 质膜蛋白的极性分布在许多细胞中发生改变， 疾病状态。 G蛋白亚基定位的定义机制 这将进一步加深我们对正常细胞反应的理解， 病态的国家

Guanine nucleotide binding proteins (G proteins) are ubiquitous proteins that transduce signals from ion channels, growth factors, hormone and neurotransmitters to a variety of intracellular effectors. In-vitro reconstitution studies with purified receptor, G protein and effector suggest that several different G proteins can interact with receptors and effectors. Other mechanisms play a role in-vivo to restrict the range of possible interactions. One way this may be achieved is by localizing the G protein to a specific plasma membrane domain. Many cells including epithelial cells segregate plasma membrane proteins (including some G proteins) to distinct surfaces. The broad long term objectives of this proposal are to define the mechanisms utilized by epithelial cells to localize G protein subunits to specific plasma membrane domains and to define the structural requirements of alpha subunits necessary for specific membrane targeting. The goals are to develop a cell culture model for the study of specific membrane localization. alphai2 localizes basolaterally in both MDCK and LLC-PK1 cells and will be labeled with an epitope from an unrelated protein to distinguish transfected from endogenous subunits. In-vitro characterization of the tagged alphai2 subunit will allow alpha subunit properties including interaction with beta gamma, binding of GTP, and myristoylation to be studied. Epitope tagged alpha i2 will be characterized to determine conditions for immunoprecipitation by the anti-epitope antibody. Tagged alpha i2 will be localized in cells by immunofluoresence and Western blots. Structural features of alpha necessary for proper sorting will be defined using mutations in alpha i2 based on my previous in-vitro work with alpha omicron. Amino and carboxyl terminal mutations in tagged alpha i2 and, chimeric alpha i2 proteins will be made to define these regions. The timing and pathway(s) of sorting will be defined using pulse chase labelling experiments and selective membrane purification. Immunoprecipitation studies using purified vesicles will begin to define other cellular elements involved in the sorting process. G proteins are critical to many cellular processes, and changes in G proteins have been linked to several diseases including cholera and Albright's Hereditary Osteodystrophy. Activating mutations in alpha/ s and alpha/i have been described in several human endocrine neoplasms. Polar distribution of plasma membrane proteins is altered in many disease states. Defining mechanisms of G protein subunit localization will further our understanding of cellular responses in normal and diseased states.