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
基本信息
- 批准号:6432902
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 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
项目摘要
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蛋白偶联的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,嗅觉甚至思考。 这些系统的核心成分是受体、异源三聚体GTP结合蛋白(G蛋白)和效应分子。G蛋白由α、β和γ亚基组成。α亚基具有鸟嘌呤核苷酸结合位点和固有的GT3活性。当激动剂与其受体相互作用形成复合物时,信号转导开始,该复合物能够促进GDP从G蛋白α亚基(G-α)释放,使得GTP可以结合并激活转导物。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蛋白可以被不可水解的GTP类似物不可逆地激活(即,Gpp[CH 2]p、Gpp[NH]p和GTP-γ-S)。 虽然对这些G蛋白介导的信号转导系统的各个组成部分了解很多,但关于这些蛋白质在完整细胞中的信号转导过程中如何相互作用还有很多有待发现。目前的假设是G蛋白被激动剂-受体复合物激活导致G-α从β-γ-异二聚体(G-β-γ)中解离,并且该系统的各个组分在细胞膜中彼此独立地移动。不可水解的GTP类似物降低G-α对G-β-γ的亲和力的事实已被用于支持GTP本身在原位激活G蛋白时引起亚基解离的假设。 使用表面等离子体共振光谱,我们已经表明,在溶液中,G-α对G-β-γ的亲和力随与G-α结合的鸟嘌呤核苷酸而变化。 我们已经确定,当与Gi-α结合的核苷酸分别为GDP、Gpp[CH 2]p、Gpp[NH]p或GTP-γ-S时,抑制性G蛋白(Gi)亚基的平衡结合常数为10、123、235或433 nM。 很明显,与GDP相比,不可水解的GTP类似物降低了Gi亚基彼此的亲和力,但其作用的可变性排除了声称它们代表GTP将如何影响Gi亚基亲和力的可能性。 G蛋白亚基之间的亲和力也受到环境的影响。 虽然在含有去污剂的溶液中,GTP-γ-S可以使G-α与G-β-γ解离,但我们已经证明,在细胞膜中,GTP-γ-S对刺激性G蛋白的活化不会引起亚基解离。 与G蛋白亚基一样,越来越多的证据表明,在细胞膜中,参与G蛋白介导的信号转导的其他组分形成了比以前假设的更紧密的复合物。 我们已经开始生物发光共振能量转移实验,以确定该系统的组件是否并列在膜中。 这些研究将帮助我们了解这些至关重要和普遍的信号转导系统是如何工作的,以便我们能够改善这些系统故障时发生的人类疾病的诊断和治疗。
项目成果
期刊论文数量(0)
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ROBERT VICTOR REBOIS其他文献
ROBERT VICTOR REBOIS的其他文献
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{{ truncateString('ROBERT VICTOR REBOIS', 18)}}的其他基金
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:
6290640 - 财政年份:
- 资助金额:
-- - 项目类别:
Elucidating The Structural Organization Of G-protein Cou
阐明 G 蛋白 Cou 的结构组织
- 批准号:
6661049 - 财政年份:
- 资助金额:
-- - 项目类别:
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:
6111865 - 财政年份:
- 资助金额:
-- - 项目类别:
Elucidating The Structural Organization Of G-protein Cou
阐明 G 蛋白 Cou 的结构组织
- 批准号:
6503234 - 财政年份:
- 资助金额:
-- - 项目类别:
Elucidating The Structural Organization Of G-protein Cou
阐明 G 蛋白 Cou 的结构组织
- 批准号:
7299405 - 财政年份:
- 资助金额:
-- - 项目类别:
Elucidating The Structural Organization Of G-protein Cou
阐明 G 蛋白 Cou 的结构组织
- 批准号:
7143854 - 财政年份:
- 资助金额:
-- - 项目类别:
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