[35S]GTPγS Binding as an Index of Total G-Protein and Gα-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 35S-labeled guanosine 5′-O-(3-thio)triphosphate ([35S]GTPγS) and on agonist stimulation a quasi-stable [35S]GTPγS–Gα complex forms and accumulates. Separation of [35S]GTPγS–Gα complexes from free [35S]GTPγS allows differences between basal and agonist-stimulated rates of [35S]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 [35S]GTPγS binding step, Gα-subunit-specific antibodies can be used to investigate the Gα-protein subpopulations activated by receptors by immunoprecipitation of [35S]GTPγS–Gα complexes and quantification by scintillation counting. Here, we describe a total [35S]GTPγS binding assay and a modification of this method that incorporates a Gα-specific immunoprecipitation step.
Key words[35S]GTPγS Guanine nucleotide binding protein G-protein-coupled receptor GDP Receptor–G-protein coupling Gα-protein-specific immunoprecipitation
- 11.Young, K.W., Bootman, M.D., Channing, D.R., Lipp, P., Maycox, P.R., Meakin, J., Challiss, R.A.J., and Nahorski, S.R. (2000) Lysophosphatidic acid-induced Ca2+ mobilization requires intracellular sphingosine 1-phosphate production: potential involvement of endogenous Edg-4 receptors. J. Biol. Chem. 275, 38532–38539.PubMedCrossRefGoogle Scholar
- 12.DeLapp, N.W., McKinzie, J.H., Sawyer, B.D., Vandergriff, A., Falcone, J., McClure, D., and Felder, C.C. (1999) Determination of [35S]guanosine-5′-O-(3-thio)triphosphate binding mediated by cholinergic muscarinic receptors in membranes from Chinese hamster ovary cells and rat striatum using an anti-G protein scintillation proximity assay. J. Pharmacol. Exp. Ther. 289, 946–955.PubMedGoogle Scholar
- 13.Salah-Uddin, H., Thomas, D.R., Davies, C.H., Hagan, J.J., Wood, M.D., Watson, J.M., and Challiss, R.A.J. (2008) Pharmacological assessment of M1 muscarinic acetylcholine receptor-Gq/11 protein coupling in membranes prepared from postmortem human brain tissue. J. Pharmacol. Exp. Ther. 325, 869–874.PubMedCrossRefGoogle Scholar
- 16.Kenakin, T. (1995) Agonist-receptor efficacy II: agonist trafficking of receptor signals. Trends Pharmacol Sci. 16, 199–205Google Scholar