INVESTIGATION OF HETEROTRIMERIC GUANINE NUCLEOTIDE BINDING PROTEIN ACTIVATION

异三聚鸟嘌呤核苷酸结合蛋白激活的研究

• 批准号: 6432902
• 负责人: ROBERT VICTOR REBOIS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432902
• 关键词: ADP ribosylation; G protein; adenylate cyclase; binding proteins; brain cell; chemical association; cow; enzyme activity; genetic transcription; genetic translation; guanine nucleoside; guanosine diphosphate; guanosine triphosphate; guanosinetriphosphatases; immunoprecipitation; membrane reconstitution /synthesis; nucleotide analog; protein structure; recombinant DNA

G-protein coupled signal transduction systems are responsible for receiving and processing information, thus enabling us to see, taste, smell and even to think. The core components of these systems are receptors, heterotrimeric GTP binding proteins (G proteins), and effector molecules. G proteins are composed of an alpha, beta, and gamma subunit. The alpha subunit has a guanine nucleotide binding site, and intrinsic GTPase activity. Signal transduction is initiated when an agonist interacts with its receptor forming a complex that is capable of facilitating the release of GDP from the G protein alpha subunit (G-alpha) so that GTP can bind and activate the transducer. The activated G protein subsequently regulates the activity of specific effector molecules until the GTP is hydrolyzed leading to deactivation of the G protein. G proteins can be irreversibly activated by non-hydrolyzable GTP analogs (ie. Gpp[CH2]p, Gpp[NH]p, and GTP-gamma-S). Although much is known about the individual components of these G protein-mediated signal transduction systems, there is much to be discovered about how these proteins interact during the signal transduction process in intact cells. The current hypothesis is that G protein activation by an agonist-receptor complex causes G-alpha to dissociate from the beta-gamma-heterodimer (G-beta-gamma), and that the individual components of this system move about independently of one another in the cell membrane. Consequently, signal transduction is thought to occur by a "random collision coupling mechanism".The fact that non-hydrolyzable GTP analogs reduce the affinity of G-alpha for G-beta-gamma has been used to buttress the hypothesis that GTP itself causes subunit dissociation when it activates G proteins in situ. Using surface plasmon resonance spectroscopy we have shown that in solution the affinity of G-alpha for G-beta-gamma varies with the guanine nucleotide that is bound to G-alpha. We have determined that the equilibrium binding constant for the subunits of the inhibitory G protein (Gi) is 10, 123, 235, or 433 nM when the nucleotide bound to Gi-alpha is GDP, Gpp[CH2]p, Gpp[NH]p or GTP-gamma-S respectively. It is clear that compared with GDP the non-hydrolyzable GTP analogs reduce the affinity of Gi subunits for each other, but the variability in their effects precludes claiming that they are representative of how GTP will effect Gi subunit affinity. The affinity of G protein subunits for each other is also affected by their environment. Although GTP-gamma-S can cause G-alpha to dissociate from G-beta-gamma in detergent containing solutions, we have shown that in cell membrane activation of the stimulatory G protein by GTP-gamma-S does not cause subunit dissociation. As with G protein subunits, there is increasing evidence that in cell membranes the other components involved in G protein mediated signal transduction form a more tightly associated complex than was previously hypothesized. We have begun bioluminescence resonance energy transfer experiments in order to determine if the components of this system are juxtaposed in the membrane. These studies will help us to understand how these critically important and pervasive signal transduction systems work so that we can improve the diagnosis and treatment of human diseases that occur when these systems malfunction.

G蛋白耦合信号转导系统负责接收和处理信息，从而使我们能够看到，品尝，气味甚至思考。 这些系统的核心成分是受体，异三聚体GTP结合蛋白（G蛋白）和效应分子。 G蛋白由α，β和伽马亚基组成。 α亚基具有鸟嘌呤核苷酸结合位点和内在的GTPase活性。当激动剂与其受体形成的受体相互作用时，开始信号转导，该复合物能够促进GDP从G蛋白α亚基（G-Alpha）中释放出来，从而使GTP可以结合并激活传感器。活化的G蛋白随后调节特异性效应分子的活性，直到GTP水解导致G蛋白失活。 G蛋白可以通过不可用的GTP类似物（即GPP [CH2] P，GPP [NH] P和GTP-GAMMA-S）不可逆地激活G蛋白。 尽管对这些G蛋白介导的信号转导系统的各个组件知之甚少，但关于这些蛋白在完整细胞的信号转导过程中如何相互作用还有很多发现。当前的假设是激动剂受体复合物的G蛋白激活导致G-Alpha从β-Gamma-Heterodimer（G-Beta-Gamma）中解离，并且该系统的个体组成部分在细胞膜中独立于彼此的彼此移动。因此，认为信号转导通过“随机碰撞耦合机制”发生。这一事实是，不可用的GTP类似物减少了G-Alpha对G-Beta-Gamma的亲和力，以支持GTP本身会引起GTP本身在激活G蛋白时引起亚基解散的假设。 使用表面等离子体的共振光谱法我们已经表明，在溶液中，G-α对G-Beta-gamma的亲和力随与G-α的鸟嘌呤核苷酸不同。 我们已经确定抑制性G蛋白（GI）亚基的平衡结合常数分别为10、123、235或433 nm，当时与GI-Alpha结合的核苷酸分别为GDP，GPP [CH2] P，GPP [NH] P或GTP-GAMMA-SP或GTP-GAMMA-S-s。 显然，与GDP相比，不可用的GTP类似物降低了GI亚基的亲和力彼此的亲和力，但是其效果的可变性排除了声称它们代表GTP将如何影响GI Subunit亲和力。 G蛋白亚基对彼此的亲和力也受其环境的影响。 尽管GTP-GAMMA-S可以在含有溶液的洗涤剂中导致G-Alpha从G-Beta-Gamma中解离，但我们已经表明，在GTP-GAMMA-S中，GTP-GAMMA-S的细胞膜激活在细胞膜激活中不会引起亚基分离。 与G蛋白亚基一样，有越来越多的证据表明，在细胞膜中，与先前假设的相关的复合物与G蛋白介导的信号转导相关的其他成分形成更紧密的复合物。 我们已经开始使用生物发光共振能量传递实验，以确定该系统的组件是否并列在膜上。 这些研究将帮助我们了解这些至关重要的和普遍的信号转导系统如何工作，以便我们可以改善这些系统故障时发生的人类疾病的诊断和治疗。