Determination of [S-35]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

An assay for measuring agonist-stimulated [S-35]guanosine-5'-O-(3-thio)trip hosphate (GTP gamma(35)S) binding to heterotrimeric GTP binding proteins wa s developed for use in 96-well format using commercially available anti-G p rotein antibodies captured by anti-IgG-coated scintillation proximity assay beads. Use of an anti-G alpha q/11 antibody to measure GTP gamma(35)S bind ing mediated by M-1, M-3, and M-5 receptors stably expressed in Chinese ham ster 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 M-1 cells before membrane preparation resulted in a m arked reduction in agonist-stimulated GTP gamma(35)S binding to whole membr anes. Direct coupling of M-1 receptors in CHO cells to inhibitory G protein s was demonstrated using an anti-G alpha i(1-3) antibody, and this binding was inhibited by 76% following PTX treatment. However, PTX had no effect on M-1-mediated binding determined using anti-G alpha q/11. CHO M-2 receptors mediated robust agonist-stimulated GTP gamma(35)S binding measured with an ti-G alpha i(1-3), but coupled only weakly to G alpha q/11. Using membranes from rat striatum, GTP gamma(35)S binding stimulated by oxotremorine M was demonstrated using anti-G alpha q/11, anti-G alpha i(1-3), and anti-G alph a o antibodies. Agonist-stimulated binding to striatal membranes showed a m arked antibody-dependent GDP requirement with robust signals obtained using 0.1 mu M GDP for anti-G alpha q/11 compared with 50 mu M GDP for anti-G al pha i(1-3) and anti-G alpha o. The potencies observed for pirenzepine and A FDX 116 blockade of agonist-stimulated GTP gamma(35)S binding to striatal m embranes determined with anti-G alpha q/11 and anti-G alpha o suggested med iation of these responses primarily by M-1 and M-4 receptors, respectively. Antibody capture GTP gamma(35)S binding using scintillation proximity assa y technology provides a convenient, productive alternative to immunoprecipi tation for exploration of receptor-G protein interaction in cells and tissu es.