Activation of Guanosine 5′-[γ-35S]thio-triphosphate Binding through M1 Muscarinic Receptors in Transfected Chinese Hamster Ovary Cell Membranes: 2. Testing the “Two-States” Model of Receptor Activation
- Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles, Brussels, Belgium
Abstract
I suggested in the accompanying article [Mol Pharmacol2001;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 ofG
from the (unstable) HRG
complex. The two effects facilitated [35S]GTPγS binding in the presence of GDP, but could be studied separately by comparing [35S]GTPγS 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 M1 muscarinic agonists' efficacy in accelerating [35S]GTPγS 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
Gi/o proteins) did not affect the agonists' efficacy profile (evaluated in the absence of spare receptors), but the addition of
GTP to the [35S]GTPγS 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”).
Footnotes
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Send reprint requests to: Dr. Waelbroeck, Laboratoire de Chimie Biologique et de la Nutrition, Faculté de Médecine de l'Université Libre de Bruxelles, Bât. G/E, CP 611, 808 Route de Lennik, B–1070 Brussels, Belgium. E-mail: mawaelbr{at}ulb.ac.be
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↵1 Because of the length of the Appendix, it is not printed herein. It can be found in its entirety in the online version of this article.
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Supported by Grant 3.4504.99 from the Fonds de la Recherche Scientifique Médicale, by an “Action de Recherche Concertée” from the “Communauté Française de Belgique” and by a “Interuniversity Poles of Attraction Program - Belgian State, Prime Minister's Office - Federal Office for Scientific, Technical and Cultural Affairs”.
- Abbreviations:
- GPCR
- G protein-coupled receptors
- GTPγS
- guanosine 5′-thio-triphosphate
- 4-DAMP mustard
- [4-diphenylacetoxy-1-(2-chloroethyl) piperidine]
- [3H]NMS
- l-[N-methyl-3H]scopolamine methyl chloride
- CHO
- Chinese hamster ovary cells
- IP
- inositol phosphate
- DMEM
- Dulbecco's minimum essential medium
- G
- GTP-bound G proteins
- G
- GTPγS-bound G proteins
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- Received August 8, 2000.
- Accepted January 9, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
