Coupling of muscarinic m1, m2 and m3 acetylcholine receptors, expressed in Chinese hamster ovary cells, to pertussis toxin-sensitive/insensitive guanine nucleotide-binding proteins.
Chinese hamster ovary (CHO) cells expressing recombinant human m1 (CHO-m1 cells), m2 (CHO-m2 cells), or m3 (CHO-m3 cells) muscarinic receptors were characterised pharmacologically with [3H]N-methylscopolamine. Agonist-stimulated coupling of these receptors with guanine nucleotide-binding proteins (G proteins) was measured by guanine nucleotide- and pertussis toxin-modification of carbachol competition-binding curves, and pertussis toxin-sensitivity of agonist-stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) binding, in membrane preparations of the CHO cell clones. High affinity agonist binding and agonist-stimulated [35S]GTP gamma S binding was abolished in CHO-m2 cell membranes (expressing 99 +/- 25 fmol of [3H]N-methylscopolamine binding sites/mg protein) after pertussis toxin pretreatment of cells, suggesting that muscarinic m2 receptors expressed in these cell membranes couple predominantly with pertussis toxin-sensitive G proteins. CHO-m1 (713 +/- 102 fmol/mg protein) and CHO-m3 (1212 +/- 279 fmol/mg protein) cell membranes produced smaller elevations in agonist-stimulated [35S]GTP gamma S binding considering the higher receptor levels, compared with CHO-m2 cell membranes. Pertussis toxin pretreatment of these clones also resulted in a significant attenuation of agonist-stimulated [35S]GTP gamma S binding suggesting that, under these experimental conditions, muscarinic m1 and m3 receptors can couple with both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. Guanine nucleotide-modification of agonist binding in CHO-m1 and CHO-m3 cell membranes was comparatively smaller than in CHO-m2 cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Burford
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NT
Department of Cell Physiology and Pharmacology, University of Leicester, UK.
Tobin
A B
AB
Nahorski
S R
SR
D000818:Animals; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D010053:Ovary / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D012601:Scopolamine / Q000494:pharmacology; D014162:Transfection; D014917:Whooping Cough
D011976:Receptors, Muscarinic; D002217:Carbachol; D012601:Scopolamine; D019204:GTP-Binding Proteins