INVESTIGATION OF HETEROTRIMERIC GUANINE NUCLEOTIDE BINDING PROTEIN ACTIVATION

G蛋白偶联信号转导系统负责接收和处理信息，从而使我们能够看、尝、闻，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部位和固有的GTP酶活性。当激动剂与其受体相互作用时，信号转导被启动，该复合体能够促进G蛋白α亚单位(G-α)释放GDP，从而使GTP结合并激活转导。激活的G蛋白随后调节特定效应分子的活性，直到GTP被水解，导致G蛋白失活。G蛋白可被不可水解的GTP类似物(即，GPP[CH2]p、GPP[NH]p和GTP-伽马-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，但对于这些蛋白如何在完整细胞的信号转导过程中相互作用仍有许多有待发现的地方。目前的假设是，由激动剂-受体复合体激活的G蛋白导致G-α从β-伽马-异二聚体(G-β-伽马)解离，并且该系统的各个组成部分在细胞膜上相互独立地移动。因此，信号转导被认为是通过“随机碰撞耦合机制”发生的。不可水解性的GTP类似物降低了G-α与G-β-γ的亲和力，这一事实被用来支持这样的假设，即GTP本身在激活G蛋白时会引起亚单位解离。利用表面等离子体共振谱，我们证明了在溶液中，G-α与G-β-γ的亲和力随与G-α结合的鸟嘌呤核苷酸的不同而不同。我们测定了抑制性G蛋白(GI)亚基的平衡结合常数为10、123、235或433 nm，分别为Gdp、GPP[CH2]p、GPP[NH]p或GTP-γ-S。显然，与GDP相比，非水解性GTP类似物降低了GI亚单位之间的亲和力，但它们作用的可变性排除了它们代表GTP如何影响GI亚单位亲和力的说法。G蛋白亚基之间的亲和力也受环境的影响。虽然在含有洗涤剂的溶液中，GTP-γ-S可以引起G-α和G-β-γ的解离，但我们已经证明，在细胞膜上，GTP-γ-S激活刺激性G蛋白不会引起亚单位解离。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成分形成了比先前假设更紧密的复合体。我们已经开始了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极其重要和普遍的信号转导系统是如何工作的，以便我们能够改进这些系统发生故障时发生的人类疾病的诊断和治疗。

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.