Activation of guanosine 5'-[gamma-(35)S]thio-triphosphate binding through M(1) muscarinic receptors in transfected Chinese Hamster ovary cell membranes: 2. Testing the "two-states" model of receptor activation.
I suggested in the accompanying article [Mol Pharmacol 2001;59:875-885] that muscarinic receptors catalyzed G protein activation. Acetylcholine or carbamylcholine recognition facilitated not only the GDP release from receptor-coupled inactive G proteins but also the release of G from the (unstable) HRG complex. The two effects facilitated [(35)S]GTP gamma S binding in the presence of GDP, but could be studied separately by comparing [(35)S]GTP gamma S binding in the absence and presence of GTP. Guanyl nucleotides affected the efficiency of receptor-G protein coupling. The relative efficacies of partial agonists in the absence and presence of GTP should remain nonlinearly correlated if all agonists stabilize (to different extents) the same active receptor conformation. The correlation between M(1) muscarinic agonists' efficacy in accelerating [(35)S]GTP gamma S binding in the absence of other nucleotides and their in vivo efficacy (inositol phosphate accumulation) was in fact very poor. This probably reflected the presence of GTP in intact cells: pertussis toxin pretreatment (which inactivates the G(i/o) proteins) did not affect the agonists' efficacy profile (evaluated in the absence of spare receptors), but the addition of GTP to the [(35)S]GTP gamma S binding medium did. These results did not support the allosteric "two states" model of receptor activation, but suggested that different agonists induced different receptor conformations ("induced fit").
Waelbroeck
M
M
Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles, Brussels, Belgium. mawaelbr@ulb.ac.be
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism* / Q000493:pharmacokinetics; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000037:antagonists & inhibitors; D008954:Models, Biological; D018721:Muscarinic Agonists / Q000378:metabolism / Q000494:pharmacology; D037342:Pertussis Toxin; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D013462:Sulfur Radioisotopes; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D018721:Muscarinic Agonists; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic; D013462:Sulfur Radioisotopes; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins