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 [H-3]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 [S-35]guanosine 5'-O-(3-thiotriphosphate) ([S-35]GTP gamma S) binding, in membrane preparations of the CHO cell clones. High affinity agonist binding and agonist-stimulated [S-35]GTP gamma S binding was abolished in CHO-m2 cell membranes (expressing 99 +/- 25 fmol of [H-3]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 [S-35]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 [S-35]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. A proportion of pertussis toxin-sensitive high affinity agonist binding sites were also detected in CHO-m3 cell membranes, but not in CHO-m1 membranes or in a CHO cell clone (CHO-vt9) expressing muscarinic m3 receptors at a lower density (430 +/- 43 fmol/mg protein) than in CHO-m3 cells. These data suggest that high affinity agonist binding to phospholipase C-linked receptors may not faithfully represent the activity of these receptors coupled to G(q/11), but may also represent these receptors coupling to pertussis toxin-sensitive G proteins.