Molecular and Cellular Analysis of G Protein Function

描述（由申请人提供）：超过一千种G蛋白偶联受体（GPCR）在广泛的生物过程中发挥作用，并且它们在健康和疾病中的重要性由于它们是数百种药物的靶标而得到强调，包括抗高血压药、神经安定药、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保持信号特异性。敲除实验表明，特定的组分通常在体内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建，这表明存在大量冗余。本实验将验证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。功能性荧光野生型和显性负性G蛋白亚基将用于探测活细胞中信号传导的细胞调节，以回答以下问题：目的I。如何调节G蛋白亚基的靶向？在HEK-293细胞中，将检查不同G蛋白β-γ复合物的形成和定位及其将Galphas靶向质膜的能力。Aim II. G蛋白亚基及其受体的依赖性转运是如何调节的？在HEK-293细胞中，调节G亚基的活化依赖性运输的机制将被确定，G蛋白异源三聚体解离和再结合的空间和时间方面将被可视化，并且将研究特定的α-γ组合对受体信号传导的重要性。Aim III.在特化细胞的分化过程中，G蛋白亚基及其受体的表达、定位和复合物的形成是如何调节的？Gs定位和信号传导将在PC 12细胞的分化过程中进行检查，PC 12细胞是生长因子刺激分化的模型系统。将确定与Galphas和被D1多巴胺受体激活的Gs异源三聚体形成并相互作用的β-γ复合物的定位模式。这些拟议的研究将产生的信息和试剂，可用于阐明在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。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.