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首页> 外文期刊>Life sciences >The (35S)GTPgammaS binding assay: approaches and applications in pharmacology.
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The (35S)GTPgammaS binding assay: approaches and applications in pharmacology.

机译：（35S）GTPgammaS结合测定：药理学中的方法和应用。

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Receptors of the of seven transmembrane spanning, heterotrimeric G protein coupled family (GPCR) play crucial roles in regulating physiological functions and consequently are targets for the action of many classes of drugs. Activation of receptor by agonist leads to the dissociation of GDP from Galpha of the Galphabetagamma heterotrimer, followed by the binding of GTP to Galpha and subsequent modulation of downstream effectors. The G protein heterotrimer is reformed by GTPase activity of the Galpha subunit, forming Galpha-GDP and so allowing Galpha and Gbetagamma to recombine. The [35S]GTPgammaS assay measures the level of G protein activation following agonist occupation of a GPCR, by determining the binding of the non-hydrolyzable analog [35S]GTPgammaS to Galpha subunits. Thus, the assay measures a functional consequence of receptor occupancy at one of the earliest receptor-mediated events. The assay allows for traditional pharmacological parameters of potency, efficacy and antagonist affinity, with the advantage that agonist measures are not subjected to amplification or other modulation that may occur when analyzing parameters further downstream of the receptor. In general the assay is experimentally more feasible for receptors coupled to the abundant G(i/o) proteins. Nevertheless, [35S]GTPgammaS binding assays are used with GPCRs that couple to the G(s) and G(q) families of G proteins, especially in artificial expression systems, or using receptor-Galpha constructs or immunoprecipitation of [35S]GTPgammaS-labeled Galpha. The relative simplicity of the assay has made it very popular and its use is providing insights into contemporary pharmacological topics including the roles of accessory proteins in signaling, constitutive activity of receptors and agonist specific signaling.
机译：七个跨膜跨膜受体，异三聚体G蛋白偶联家族（GPCR）在调节生理功能中起关键作用，因此是许多类药物作用的靶标。激动剂激活受体会导致GDP与Galphabetagamma异源三聚体的Galpha分离，随后GTP与Galpha结合并随后调节下游效应子。 G蛋白异源三聚体通过Galpha亚基的GTPase活性进行重组，形成Galpha-GDP，因此允许Galpha和Gbetagamma重组。 [35S] GTPgammaS分析法通过确定不可水解的类似物[35S] GTPgammaS与Galpha亚基的结合来测量GPCR激动剂占用后G蛋白活化的水平。因此，该测定法在最早的受体介导的事件之一上测量了受体占据的功能性后果。该测定法考虑了效力，功效和拮抗剂亲和力的传统药理学参数，其优点在于，在分析受体更下游的参数时，激动剂措施不会受到扩增或其他调节的影响。通常，对于与大量G（i / o）蛋白偶联的受体，该实验在实验上更可行。不过，[35S] GTPgammaS结合测定法可用于与G蛋白的G（s）和G（q）家族偶联的GPCR，尤其是在人工表达系统中，或使用[35S] GTPgammaS-的受体-Galpha构建体或免疫沉淀标记为Galpha。该测定法的相对简单性使其非常受欢迎，其使用为当代药理学话题提供了见识，包括辅助蛋白在信号传导中的作用，受体的组成活性和激动剂特异性信号传导。

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