The [35S]GTPγS binding assay: approaches and applications in pharmacology

Abstract 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 Gα of the Gαβγ heterotrimer, followed by the binding of GTP to Gα and subsequent modulation of downstream effectors. The G protein heterotrimer is reformed by GTPase activity of the Gα subunit, forming Gα-GDP and so allowing Gα and Gβγ to recombine. The [ 35 S]GTPγS assay measures the level of G protein activation following agonist occupation of a GPCR, by determining the binding of the non-hydrolyzable analog [ 35 S]GTPγS to Gα 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, [ 35 S]GTPγS 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-Gα constructs or immunoprecipitation of [ 35 S]GTPγS-labeled Gα. 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.