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


METHODS
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.


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
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.
Jakubík
J
J
Institute of Physiology Academy of Sciences of the Czech Republic, Prague, Czech Republic. jakubik@biomed.cas.cz
Janíčková
H
H
El-Fakahany
E E
EE
Doležal
V
V
D000494:Allosteric Regulation; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019204:GTP-Binding Proteins / Q000145:classification / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D018721:Muscarinic Agonists / Q000378:metabolism*; D018727:Muscarinic Antagonists / Q000378:metabolism; D019832:N-Methylscopolamine / Q000378:metabolism; D011485:Protein Binding; D011869:Radioligand Assay; D043585:Receptor, Muscarinic M2 / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D043585:Receptor, Muscarinic M2; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D019832:N-Methylscopolamine