MOLECULAR AND CELLULAR ANALYSIS OF G PROTEIN FUNCTION

异源三聚体G蛋白将七跨膜螺旋受体接收的激素和感觉信号传递给介导各种生理过程的效应蛋白。G蛋白偶联受体是数百种药物的靶点，包括抗高血压药、抗精神病药、抗组胺药和抗抑郁药，这一事实强调了G蛋白信号通路在健康和疾病中的重要性。 一个重要的挑战是阐明受体和G蛋白之间的相互作用如何影响它们的活性以及这些相互作用如何在体内调节的水平上的调节。 对这些过程的进一步了解将为异常信号通路的治疗干预提供更广泛的靶点和策略。 本研究的目的是从分子水平和细胞水平阐明G蛋白信号转导的机制。 这个建议的具体目标是回答这些问题：（I）是什么调节G蛋白和受体在激活周期中的结合/解离？ 将研究α的表面暴露亚基区域的作用，其中取代增加受体亲和力。 将定位关键残基，并确定突变它们的功能效应。 将检测该区域中的α残基，以确定其是否可赋予β 2-肾上腺素能受体特异性。 将测试基于该区域的肽，以观察它们是否模拟或阻断Gs与β 2-肾上腺素能受体之间的相互作用。(II)功能不同的受体群体能被显性负性α亚基选择性失活吗？ 既增加受体亲和力又降低效应子亲和力的突变将组合以产生强显性负性α亚基。显性负性α亚基将用于阐明与多种G蛋白偶联的受体的生理作用。(III)受体和G蛋白的细胞组织如何调节信号传导？ 关于G蛋白介导的信号转导的大部分已知是基于使用纯化组分的生化实验和固定细胞中蛋白质的免疫组织化学定位的静态图像。为了确定这些结论可以扩展到体内情况的程度，将在活细胞中使用延时荧光显微镜、光漂白后荧光恢复、荧光共振能量转移和荧光相关光谱法来跟踪与可区分的GFP突变体融合的蛋白质对（α/β、α/受体、B/受体）。

Heterotrimeric G proteins transmit hormonal and sensory signals received by seven-transmembrane-helix receptors to effector proteins that mediate a wide variety of physiological processes. The importance of G protein signaling pathways in health and disease is underscored by the fact that G protein-coupled receptors are the targets of hundreds of drugs, including antihypertensives, neuroleptics, antihistamines, and antidepressants. An important challenge is to elucidate regulation at the level of how interactions between receptors and G proteins affect their activities and how these interactions are regulated in vivo. An increased understanding of these processes will provide a wider range of targets and strategies for therapeutic interventions for aberrant signaling pathways. The goal of this proposal is to elucidate the mechanism of G protein signaling at both the molecular and cellular levels. The specific aims of this proposal are to answer these questions: (I) What regulates association/dissociation of G proteins and receptors during the activation cycle? The role of a surface- exposed subunit region of alphas in which substitutions increase receptor affinity will be investigated. The critical residues will be localized and the functional effects of mutating them will be determined. Alphas residues in this region will be tested to see if they can confer specificity for the Beta2- adrenergic receptor. Peptides based on this region will be tested to see if they mimic or block interactions between Gs and the Beta2-adrenergic receptor. (II) Can functionally distinct receptor populations be selectively inactivated by dominant negative alpha subunits? Mutations that both increase receptor affinity and decrease effector affinity will be combined in an effort to produce strong dominant negative alpha subunits. Dominant negative alpha subunits will be used to clarify the physiological roles of receptors that couple to multiple G proteins. (III) How does the cellular organization of receptors and G proteins regulate signaling? Much of what is known about G protein-mediated signal transduction is based on biochemical experiments using purified components and static images of immunohistochemical localization of the proteins in fixed cells. To determine the extent to which these conclusions can be extended to in vivo situations, pairs of proteins (alpha/beta, alpha/receptor, b/receptor) fused to distinguishable GFP mutants will be followed in living cells using time lapse fluorescence microscopy, fluorescence recover after photobleaching, fluorescence resonance energy transfer, and fluroescence correlation spectroscopy.