Activation of Guanosine 5′-[γ-35S]thio-triphosphate Binding through M1 Muscarinic Receptors in Transfected Chinese Hamster Ovary Cell Membranes: 1. Mathematical Analysis of Catalytic G Protein Activation
- Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles, Brussels, Belgium
Abstract
I analyzed in this work the effect of agonists and unlabeled guanyl nucleotides on [35S]GTPγS and [3H]NMS binding to transfected CHO cells expressing hM1 muscarinic receptors. I was unable to explain my kinetic results by “traditional” (one-site, two-site, or two-step) bimolecular
binding models. I therefore examined the equations that describe catalytic G protein activation. My results were fully consistent
with the following interpretation: G protein-coupled receptors either interacted with GDP-bound G proteins and facilitated
the GDP release or recognized empty G proteins, depending on the incubation conditions. The receptor-coupled empty G proteins
(RG) then recognized GTPγS, and the occupied G protein (G
) dissociated reversibly from the receptor. Agonists accelerated the GDP release from receptor-coupled G proteins and accelerated
theG
dissociation: both effects accelerated synergically the G protein-GTPγS association reaction in the presence of GDP. GTPγS-bound
G proteins,G
, competed efficiently with inactive (empty or GDP-bound) G proteins for receptor recognition, and were able, therefore, at
low concentrations, to quench the [35S]GTPγS binding reaction.
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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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”.
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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.
- Abbreviations:
- GPCR
- G protein-coupled receptors
- GTPγS
- guanosine 5′-thio-triphosphate
- CHO
- Chinese hamster ovary cells
- 4-DAMP mustard
- [4-diphenylacetoxy-1-(2-chloroethyl) piperidine]
- [3H]NMS
- l-[N-methyl-3H]scopolamine methyl chloride
- 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
