Molecular and Cellular Analysis of G Protein Function

G 蛋白功能的分子和细胞分析

• 批准号: 7252067
• 负责人: CATHERINE H BERLOT
• 金额: $ 25.91万
• 依托单位: GEISINGER CLINIC
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7252067
• 关键词: Accounting; Address; Adrenergic Agents; Adrenergic Receptor; Affect; Agonist; Animal Model; Antidepressive Agents; Antihistamines; Antihypertensive Agents; Antipsychotic Agents; Arrestin; Arrestins; Binding; Biological Assay; Biological Models; Biological Process; Calcitonin; Calcitonin Receptor; Catalytic RNA; Cell Differentiation process; Cell membrane; Cells; Cellular Structures; Complex; Couples; Cytoskeleton; Data; Disease; Dissociation; Dominant-Negative Mutation; Dopamine; Dopamine D1 Receptor; Dopamine Receptor; Endocytosis; Exhibits; Fluorescence; Fluorescence Recovery After Photobleaching; Fluorescence Resonance Energy Transfer; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gays; Green Fluorescent Proteins; Growth Cones; Growth Factor; Health; Hormones; Image; In Vitro; Intracellular Membranes; Knock-out; Life; Localized; Measures; Mediating; Membrane Microdomains; Modeling; Molecular; Neuronal Differentiation; PC12 Cells; Pathway interactions; Pattern; Pharmaceutical Preparations; Phosphorylation; Play; Protein Isoforms; Protein Subunits; Proteins; Range; Reagent; Receptor Signaling; Research Personnel; Role; Role playing therapy; Signal Pathway; Signal Transduction; Signaling Protein; Site; Specificity; Testing; Tissues; Vesicle; Work; Yin; adrenergic; base; cell growth regulation; cell type; design; dimer; dopamine D5 receptor; in vivo; mutant; neurite growth; neuronal cell body; preference; programs; protein expression; protein function; receptor; receptor internalization; reconstitution; research study; response; trafficking

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.

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，它们在健康和疾病中的重要性被数百种药物的靶标所强调，包括抗高血压药、神经抑制剂、抗组胺药和抗抑郁药。一个重要的，但知之甚少的问题是信号特异性是如何在体内维持的。基因敲除实验表明，特定组分在体内通常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，这表明存在大量冗余。本课题的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。功能荧光野生型和显性负G蛋白亚基将用于探索活细胞中信号的细胞调控，以回答以下问题：目的1 . G蛋白亚基的靶向性是如何调节的？在HEK-293细胞中，我们将研究不同G蛋白- β - γ复合物的形成和定位，以及它们将Galphas靶向质膜的能力。目的二世。G蛋白亚基及其受体的激素依赖性运输是如何调控的？在HEK-293细胞中，将确定Gs亚基激活依赖性运输的调节机制，G蛋白异源三聚体解离和重新结合的时空方面将被可视化，并研究特异性字母表组合对受体信号传导的重要性。第三目标。在特化细胞分化过程中，G蛋白亚基及其受体的表达、定位和复杂形成是如何调控的？在PC12细胞的分化过程中，Gs的定位和信号将被检查，PC12细胞是一个生长因子刺激分化的模型系统。形成并与Galphas和被D1多巴胺受体激活的Gs异源三聚体相互作用的β - γ复合物的定位模式将被确定。这些拟议的研究将产生信息和试剂，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。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。