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Br J Pharmacol. 2011 Mar;162(5):1029-44. doi: 10.1111/j.1476-5381.2010.01081.x.

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

Author information

  • 1Institute of Physiology Academy of Sciences of the Czech Republic, Prague, Czech Republic. jakubik@biomed.cas.cz

Abstract

BACKGROUND AND PURPOSE:

Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy.

EXPERIMENTAL APPROACH:

Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor.

KEY RESULTS:

Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins.

CONCLUSIONS AND IMPLICATIONS:

These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding.

© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

PMID:
20958290
[PubMed - indexed for MEDLINE]
PMCID:
PMC3051377
Free PMC Article
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