G PROTEIN-COUPLED RECEPTORS--STRUCTURE AND REGULATION

G 蛋白偶联受体——结构和调控

• 批准号: 2021900
• 负责人: Elliott M Ross
• 金额: $ 37.98万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2021900
• 关键词: G protein; L cell; MDCK cell; PC12 cells; Sf9 cell line; alpha adrenergic receptor; beta adrenergic receptor; chimeric proteins; crystallization; endocytosis; fluorescent dye /probe; glutamate receptor; guanosine triphosphate; guanosinetriphosphatases; high performance liquid chromatography; hydrogen; immunocytochemistry; ionophores; laboratory mouse; laboratory rabbit; membrane model; membrane transport proteins; muscarinic receptor; mutant; northern blottings; phospholipase C; protein engineering; protein purification; protein structure function; receptor coupling; sodium

I propose to study the mechanism of regulation of G proteins by receptors; the structural determinants of receptor function, specificity and regulation; and the organization of G protein-mediated signaling pathways. (1) We will investigate the mechanism of action of a cytoplasmic C-terminal domain that alters several aspects of receptors' activity and regulation, probably by anchoring receptor to a fixed structure at or on the plasma membrane. We will evaluate similar domains on other receptors and attempt to purify the protein(s) to which this domain binds. (2) We recently found that the m1 muscarinic receptor regulates phospholipase C activity through Gq and that PLC-beta1 promotes the deactivation of Gq by stimulating the GTPase activity of Gq. We will characterize this feedback regulation in receptor-Gq-PLC vesicles and relate it kinetically to the overall activation of the Gq pathway. We will also determine if other G protein-regulated effectors have GTPase- stimulating activity. (3) We will continue to study the mechanisms by which receptors selectively regulate specific G proteins. We will define the important structural features of the selectivity-determining domains that we have identified in the receptors' second and third cytoplasmic loops. We will use defined, recombinant betagamma subunit dimers purified from Baculovirus-infected Sf9 cells to analyze the contribution of individual beta and gamma subunits to receptor-G protein selectivity. (4) We will evaluate the extent of Gs-independent signaling by the beta- adrenergic receptor in Gs-deficient cyc- cells and explore its mechanism. We will determine the pathway by which this receptor stimulates the Na+/H+ exchanger, a widespread beta-adrenergic response that does not require Gs or cyclic AMP. We will focus on whether stimulation is direct or second messenger-mediated. We will evaluate the ability of the receptor to regulate Gq and Gi both in cyc- cells and in vitro. Receptor-Gq coupling will be evaluated as a possible mechanism for beta-adrenergic regulation of the exchanger. (5) We will continue studying mutations of the beta-adrenergic receptor that improves its expression. We will collaborate with Richard Henderson on the receptor's large scale purification, stabilization and crystallization.

我建议研究受体调节G蛋白的机理； 受体功能，特异性和 规定;以及G蛋白介导的信号通路的组织。 （1）我们将研究细胞质C末端的作用机理 改变受体活性和调节的几个方面的领域， 可能是通过将受体锚定在等离子体的固定结构上 膜。 我们将评估其他受体上的类似领域并尝试 纯化该结构域结合的蛋白质。 （2）我们最近发现M1毒蕈碱受体调节 通过GQ的磷脂酶C活性，PLC-BETA1促进了 通过刺激GQ的GTPase活性，将GQ失活。 我们将 表征受体-GQ-PLC囊泡中的反馈调节和 将其与GQ途径的整体激活相关。 我们将 还确定其他G蛋白调节的效应子是否具有GTPase- 刺激活动。 （3）我们将继续研究受体选择性的机制 调节特定的G蛋白。 我们将定义重要的结构 我们已经确定的选择性确定域的特征 受体的第二和第三个细胞质环。 我们将使用定义的 重组的Betagamma亚基二聚体从杆状病毒感染的SF9纯化 细胞分析单个β和伽马亚基对 受体-G蛋白选择性。 （4）我们将评估β- GS缺陷CYC细胞中的肾上腺素能受体并探索其机制。 我们将确定该受体刺激Na+/H+的途径 Exchanger，一种不需要GS的广泛β-肾上腺素能反应 或循环放大器。 我们将专注于刺激是直接还是第二 Messenger介导的。 我们将评估受体的能力 在CYC细胞和体外调节GQ和GI。 受体-GQ耦合 将评估作为β-肾上腺素能调节的可能机制 交换器。 （5）我们将继续研究β-肾上腺素能受体的突变 这改善了其表达。 我们将与理查德·亨德森合作 在受体的大规模纯化，稳定和 结晶。