Abstract
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.
Footnotes
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Send reprint requests to: Neil W. DeLapp, Lilly Corporate Center, Indianapolis, IN 46285. E-mail:NWD{at}lilly.com
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↵1 A preliminary account of this work was presented at the Eighth International Subtypes of Muscarinic Receptors Meeting.
- Abbreviations:
- AFDX 116
- 11-{[2-((diethylamino)methyl)-1-piperidinyl]acetyl}-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one
- CHO
- Chinese hamster ovary cells
- PTX
- pertussis toxin
- SPA
- scintillation proximity assay
- WGA
- wheat germ agglutinin
- Received October 22, 1998.
- Accepted December 30, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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