Coupling of muscarinic m1, m2 and m3 acetylcholine receptors, expressed in Chinese hamster ovary cells, to pertussis toxin-sensitive/insensitive guanine nucleotide-binding proteins

Abstract

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]GTPγS) binding, in membrane preparations of the CHO cell clones. High affinity agonist binding and agonist-stimulated [ 35S]GTP-γS binding was abolished in CHO-m2 cell membranes (expressing 99 ± 25 fmol of [ 3 H]N- methylcopolamine 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 maller elevations in agonist-stimulated [ 35S]GTPγS binding considering the higher receptor levels, compared with CHO-m2 cell membranes. Pertussis toxin pretreatment of these clones clso resulted in a significant attenuation of agonist-stimulated [ 35S]GTPγS 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.