Assay of G protein-coupled receptor activation of G proteins in native cell membranes using [35S]GTP gamma S binding.

The interaction of a G protein-coupled receptor (GPCR) with the G protein is the first step in the transduction of receptor binding to the activation of second-messenger systems mediated by G proteins. Binding of the poorly hydroylzable GTP analog guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]-GTP gamma S) to the alpha subunit of a G protein has been used as a biochemical assay to measure the efficacy of a compound. The maximal percent stimulation of [35S]GTP gamma S binding, induced by an agonist, correlates well with the efficacy of an agonist in an in vivo system. The concentration of agonist necessary to achieve 50% of the maximal stimulation is indicative of the affinity of the agonist for the receptor, under the assay conditions. The [35S]GTP gamma S binding assay, which uses membranes from either myeloma cells or endogenous tissues, is a biochemical assay to determine the efficacies of agonists for receptors that are expressed endogenously. Novel compounds that have been shown to have high affinity in radioligand receptor-binding assays are screened in the [35S]GTP gamma S binding assay to determine if they are agonists, antagonists, or partial agonists at a particular receptor.