Molecular and Cellular Analysis of G Protein Function

描述(申请人提供)：一千多个G蛋白偶联受体(GPCRs)在广泛的生物过程中发挥作用，它们在健康和疾病中的重要性因它们是数百种药物的靶标这一事实而得到强调，包括抗高血压药、神经镇静剂、抗组胺药和抗抑郁药。一个重要但知之甚少的问题是如何在体内维持信号的特异性。基因敲除实验表明，特定的成分在体内往往具有不可或缺的作用，但许多途径和相互作用可以在体外重组，这表明存在大量的冗余。该方案中的实验将检验信号蛋白的细胞定位和组织在特异性中起重要作用的假设。功能荧光野生型和显性负性G蛋白亚基将被用来探索活细胞中信号的细胞调控，以回答以下问题：目的I G蛋白亚基的靶向是如何调节的？在HEK-293细胞中，将检测不同G蛋白β-淀粉酶复合体的形成和定位，以及它们将GalpHa靶向质膜的能力。目的II.激素依赖的G蛋白亚基及其受体的运输是如何调节的？在HEK-293细胞中，将确定调控Gs亚单位激活依赖的运输的机制，将可视化G蛋白异三聚体解离和重新解离的空间和时间方面，并将研究特定的α-配基组合对受体信号传递的重要性。目的III.在特化细胞分化过程中，G蛋白亚基及其受体的表达、定位和复合体的形成是如何调节的？GS的定位和信号将在PC12细胞的分化过程中进行检测，PC12细胞是生长因子刺激分化的模型系统。将确定与D_1多巴胺受体激活的Galpas和Gs异三聚体形成并相互作用的Betagamma复合体的定位模式。这些拟议的研究将产生可用于阐明各种细胞类型和动物模型中G蛋白信号特异性的分子和细胞基础的信息和试剂。最终，这些方法将有助于设计策略来操纵导致疾病的异常信号通路。

DESCRIPTION (provided by applicant): More than a thousand G protein-coupled receptors (GPCRs) play roles in a vast range of biological processes and their importance in health and disease is underscored by the fact that they are the targets of hundreds of drugs, including antihypertensives, neuroleptics, antihistamines, and antidepressants. An important, but poorly understood issue is how signaling specificity is maintained in vivo. Knockout experiments have shown that particular components often have indispensable roles in vivo, but many pathways and interactions can be reconstituted in vitro, suggesting a large amount of redundancy. The experiments in this proposal will test the hypothesis that the cellular localization and organization of signaling proteins play an important role in the specificity. Functional fluorescent wild-type and dominant negative G protein subunits will be used to probe the cellular regulation of signaling in living cells in order to answer the following questions: Aim I. How is targeting of G protein subunits regulated? In HEK-293 cells, the formation and localization of different G protein betagamma complexes and their abilities to target Galphas to the plasma membrane will be examined. Aim II. How are hormone-dependent trafficking of G protein subunits and their receptors regulated? In HEK-293 cells, the mechanisms that regulate activation-dependent trafficking of the subunits of Gs will be determined, the spatial and temporal aspects of G protein heterotrimer dissociation and reassociation will be visualized, and the importance of specific alphabetagamma combinations for receptor signaling will be investigated. Aim III. How are expression, localization, and complex formation of G protein subunits and their receptors regulated during differentiation of a specialized cell? Gs localization and signaling will be examined during differentiation of PC12 cells, a model system for growth-factor stimulated differentiation. The localization patterns of betagamma complexes that form and interact with Galphas and the Gs heterotrimers that are activated by the D1 dopamine receptor will be determined. These proposed studies will produce information and reagents that can be applied to elucidate the molecular and cellular basis for G protein signaling specificity in a variety of cell types and animal models. Ultimately, these approaches will facilitate the design of strategies to manipulate aberrant signaling pathways responsible for disease.