FUNCTION AND REGULATION OF G PROTEIN COUPLED RECEPTORS

G蛋白偶联受体控制着多种生理性的 是药物干预的潜在靶点。 无数的疾病。有成百上千的受体介导 对激素和神经递质的反应以及对不同 刺激物，如光、气味和化学诱导剂。然而，所有这些 受体表现出共同的拓扑结构，它们都可以转导 信号由相同的基本过程控制，许多信号可能受 相似的细胞内途径。了解以下机制： 激活这些重要的分子开关和发现 控制其活性的相关蛋白质是杰出的 未来生物医学研究的问题。 遗传学、生物物理学和细胞生物学方法的结合， 应用于盘状葡萄球菌的cAMP化学吸引受体(CARS)， 旨在阐明G-蛋白的功能和调节 一般情况下是偶联受体。体质活动性和过敏性 受体将通过随机突变和表型分离 放映。纯化的重组cAR1显示出强大的激动剂诱导 内源性色氨酸荧光减弱，可能表明 全球构象变化。CAMP结合动力学的研究， 激动剂诱导的形状变化，以及 带有G蛋白的纯化受体被设计用于研究 激活。选定色氨酸的替代和附着物 将使用针对特定半胱氨酸和赖氨酸残基的荧光探针 探讨激动剂诱导运动的局部结构域。至 描绘了受体兴奋的各个阶段，这些纯化的研究 蛋白质将包括一组携带点突变的受体 已经在体内被证明可以降低亲和力，防止激活，引起 结构性激活，或稳定高亲和力中间体 州政府。一个长期目标是确定受体的结构。 在存在和不存在激动剂的情况下的受体/G蛋白复合体 在二维和三维晶体中。CAR1-GFP是一致的 分布在单个活细胞的膜上。侧向 化学上前脑和脑后受体的流动性 定向细胞和不同磷酸化受体的 下定决心。激动剂诱导的cAR1磷酸化降低其 亲和力，但与教义相反，磷酸化不是必需的 反应终止或趋化。发现涉及的新途径 在脱敏过程中，筛选带有基因标签的细胞系 对持续刺激的持续反应是有计划的。一种基因， 代号，CAMP-抗性A，已被分离并生化 缺乏该基因的细胞规避脱敏的机制 正在接受调查。

G-protein coupled receptors control a immense variety of physiological responses and are potential targets for pharmacological intervention in numerous diseases. There are hundreds of receptors that mediate responses to hormones and neurotransmitters as well as to diverse stimuli such as light, odorants, and chemoattractants. Yet all of these receptors display a common topological structure, they may all transduce signals by the same basic processes, and many may be regulated by similar intracellular pathways. Understanding the mechanisms of activation of these important molecular switches and discovery of associated proteins that control their activities are outstanding questions for future biomedical research. A combination of genetic, biophysical, and cell biological approaches, applied to the cAMP chemoattractant receptors (cARs) in D. discoideum, are intended to elucidate the function and regulation of G-protein coupled receptors in general. Constitutively active and hypersensitive receptors will be isolated by random mutagenesis and phenotypic screening. Purified, recombinant cAR1 displays a robust agonist-induced decrease in intrinsic tryptophan fluorescence, likely indicating a global conformational change. Studies of the kinetics of cAMP binding, of the agonist-induced shape changes, and of the interactions of the purified receptors with G-proteins are designed to investigate activation. Substitution of selected tryptophans and attachment of fluorescent probes to specific cysteine and lysine residues will be used to explore local domains involved in the agonist-induced movements. To delineate the stages of receptor excitation, these studies of purified proteins will include a panel of receptors bearing point mutations that have been shown in vivo to decrease affinity, prevent activation, cause constitutive activation, or stabilize a high-affinity intermediate state. A long term goal is to determine the structure of the receptors and receptor/G-protein complexes in the presence and absence of agonists in two- and three-dimensional crystals. cAR1-GFP is uniformly distributed along the membrane of single living cells. The lateral mobility of receptors at the fronts and backs of chemotactically oriented cells and of differently phosphorylated receptors will be determined. Agonist-induced phosphorylation of cAR1 decreases its affinity but, contrary to dogma, phosphorylation is not required for response termination or chemotaxis. To discover novel pathways involved in desensitization, screens of gene-tagged cell lines for cells that continually respond to persistent stimulation are planned. One gene, designated, cAMPS-resistance A, has been isolated and the biochemical mechanisms by which cells lacking this gene circumvent desensitization are being investigated.