Measurement of Agonist-Stimulated [35S]GTPγS Binding to Assess Total G-Protein and Gα-Subtype-Specific Activation by G-Protein-Coupled Receptors

On activation, G-protein-coupled receptors (GPCRs) exert many of their cellular actions through promotion of guanine nucleotide exchange on the Gα-subunit of heterotrimeric G-proteins to release free Gα-GTP and βγ-subunits. In membrane preparations, GTP can be substituted by ³⁵ S-labeled guanosine 5′-O -(3-thio)triphosphate ([³⁵ S]GTγS) and on agonist stimulation a stable [³⁵ S]GTPγS-Gα complex will form and accumulate. Separation of ³⁵ S-bound 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.