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.
An assay for measuring agonist-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate (GTPgamma35S) 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-Galphaq/11 antibody to measure GTPgamma35S 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 GTPgamma35S binding to whole membranes. Direct coupling of M1 receptors in CHO cells to inhibitory G proteins was demonstrated using an anti-Galphai(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-Galphaq/11. CHO M2 receptors mediated robust agonist-stimulated GTPgamma35S binding measured with anti-Galphai(1-3), but coupled only weakly to Galphaq/11. Using membranes from rat striatum, GTPgamma35S binding stimulated by oxotremorine M was demonstrated using anti-Galphaq/11, anti-Galphai(1-3), and anti-Galphao antibodies. Agonist-stimulated binding to striatal membranes showed a marked antibody-dependent GDP requirement with robust signals obtained using 0.1 microM GDP for anti-Galphaq/11 compared with 50 microM GDP for anti-Galphai(1-3) and anti-Galphao. The potencies observed for pirenzepine and AFDX 116 blockade of agonist-stimulated GTPgamma35S binding to striatal membranes determined with anti-Galphaq/11 and anti-Galphao suggested mediation of these responses primarily by M1 and M4 receptors, respectively. Antibody capture GTPgamma35S binding using scintillation proximity assay technology provides a convenient, productive alternative to immunoprecipitation for exploration of receptor-G protein interaction in cells and tissues.
DeLapp
N W
NW
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, USA. NWD@lilly.com
McKinzie
J H
JH
Sawyer
B D
BD
Vandergriff
A
A
Falcone
J
J
McClure
D
D
Felder
C C
CC
D000465:Algorithms; D000818:Animals; D000906:Antibodies; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D051379:Mice; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D017072:Neostriatum / Q000187:drug effects / Q000378:metabolism*; D010095:Oxotremorine / Q000494:pharmacology; D010890:Pirenzepine / Q000031:analogs & derivatives / Q000494:pharmacology; D011233:Precipitin Tests / Q000379:methods; D051381:Rats; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000378:metabolism
D000906:Antibodies; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010890:Pirenzepine; D010095:Oxotremorine; D019204:GTP-Binding Proteins; C048593:otenzepad