[35S]GTPS Binding as an Index of Total G-Protein and G-Subtype-Specific Activation by GPCRs

On activation, G-protein-coupled receptors (GPCRs) exert many of their cellular actions through �promoting guanine nucleotide exchange on Gα subunits of heterotrimeric G proteins to release Gα-GTP and free βγ-subunits. In membrane preparations, GTP can be substituted by ³⁵ S-labeled guanosine� 5′-O -(3-thio)triphosphate ([³⁵ S]GTPγS) and on agonist stimulation a quasi-stable [³⁵ S]GTPγS–Gα �complex forms and accumulates. Separation of [³⁵ S]GTPγS–Gα complexes from free [³⁵ S]GTPγS allows differences between basal and agonist-stimulated rates of [³⁵ S]GTPγS–Gα complex formation� to be used to obtain pharmacological information on receptor–G-protein information transfer. Further, by releasing Gα-subunits into solution following the [³⁵ S]GTPγS binding step, Gα-subunit-specific antibodies can be used to investigate the Gα-protein subpopulations activated by receptors by immunoprecipitation of [³⁵ S]GTPγS–Gα complexes and quantification by scintillation counting. Here, we describe a total [³⁵ S]GTPγS binding assay and a modification of this method that incorporates a Gα-specific immunoprecipitation step.