PT  - JOURNAL ARTICLE
AU  - Neil W. Delapp
AU  - Jamie H. McKinzie
AU  - Barry D. Sawyer
AU  - Amy Vandergriff
AU  - Julie Falcone
AU  - Don McClure
AU  - Christian C. Felder
TI  - Determination of [<sup>35</sup>S]Guanosine-5′-<em>O</em>-(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
DP  - 1999 May 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 946--955
VI  - 289
IP  - 2
4099  - http://jpet.aspetjournals.org/content/289/2/946.short
4100  - http://jpet.aspetjournals.org/content/289/2/946.full
SO  - J Pharmacol Exp Ther1999 May 01; 289
AB  - An assay for measuring agonist-stimulated [35S]guanosine-5′-O-(3-thio)triphosphate (GTPγ35S) binding to heterotrimeric GTP binding proteins was developed for use in 96-well format using commercially available anti-G protein antibodies captured by anti-IgG-coated scintillation proximity assay beads. Use of an anti-Gαq/11 antibody to measure GTPγ35S binding mediated by M1, M3, and M5 receptors stably expressed in Chinese hamster ovary (CHO) cells resulted in a marked increase in agonist-stimulated/basal binding ratio compared with whole membrane binding. Pertussis toxin (PTX) treatment of CHO M1 cells before membrane preparation resulted in a marked reduction in agonist-stimulated GTPγ35S binding to whole membranes. Direct coupling of M1 receptors in CHO cells to inhibitory G proteins was demonstrated using an anti-Gαi(1–3) antibody, and this binding was inhibited by 76% following PTX treatment. However, PTX had no effect on M1-mediated binding determined using anti-Gαq/11. CHO M2 receptors mediated robust agonist-stimulated GTPγ35S binding measured with anti-Gαi(1–3), but coupled only weakly to Gαq/11. Using membranes from rat striatum, GTPγ35S binding stimulated by oxotremorine M was demonstrated using anti-Gαq/11, anti-Gαi(1–3), and anti-Gαo antibodies. Agonist-stimulated binding to striatal membranes showed a marked antibody-dependent GDP requirement with robust signals obtained using 0.1 μM GDP for anti-Gαq/11 compared with 50 μM GDP for anti-Gαi(1–3) and anti-Gαo. The potencies observed for pirenzepine and AFDX 116 blockade of agonist-stimulated GTPγ35S binding to striatal membranes determined with anti-Gαq/11 and anti-Gαo suggested mediation of these responses primarily by M1 and M4 receptors, respectively. Antibody capture GTPγ35S binding using scintillation proximity assay technology provides a convenient, productive alternative to immunoprecipitation for exploration of receptor-G protein interaction in cells and tissues. The American Society for Pharmacology and Experimental Therapeutics