Binding of GTPgamma[35S] is regulated by GDP and receptor activation. Studies with the nociceptin/orphanin FQ receptor.
OBJECTIVE
We have examined the effects of ligand efficacy and receptor density on the binding of guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) and GDP to the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP)-coupled G-proteins.


METHODS
In GTPgamma[(35)S] binding experiments, using stable (CHO(hNOP)) and inducible (CHO(INDhNOP)) recombinant human and rat NOP we have measured: (i) ligand-specific GDP requirements; (ii) the effects of receptor density on guanine nucleotide affinity/capacity; and (iii) the effect of ligand efficacy on GTPgammaS association kinetics.


RESULTS
GTPgammaS competition curves were shallow and modelled by high- and low-affinity components that were relatively consistent between cell types and tissue preparations. In the presence of 1 microM N/OFQ a high-affinity GDP binding site was also present, but the fraction of total binding was reduced. In an efficacy-dependent manner, the partial agonists [F/G]N/OFQ(1-13)NH(2) ([Phe(1)psi(CH(2)-NH)Gly(2)]-nociceptin(1-13)NH(2)) and naloxone benzoylhydrazone both reduced the fraction of high-affinity sites for GDP (relative to basal). While the pIC(50) for high-affinity GDP binding site did not decrease in the presence of 1 microM N/OFQ, N/OFQ produced a significant reduction in pIC(50) for the low-affinity site. Agonist-mediated decrease in affinity for GDP binding was efficacy-dependent. GDP displayed three affinities: high, conserved in the presence and absence of ligand; intermediate, present as a low fraction under basal conditions; low (efficacy-dependent), present during receptor activation representing the majority of binding.


CONCLUSIONS
The affinity of GTPgamma[(35)S] was regulated by GDP and receptor activation caused increased binding of GTPgamma[(35)S] through a reduction in GDP affinity.
Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester Royal Infirmary, Leicester, UK.
John
J
McDonald
David G
DG
Lambert
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004441:Ecdysterone / Q000031:analogs & derivatives / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000819:agonists / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008024:Ligands; D009270:Naloxone / Q000031:analogs & derivatives / Q000494:pharmacology; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D011957:Receptors, Opioid / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D000094942:Nociceptin Receptor
D008024:Ligands; D011957:Receptors, Opioid; D013462:Sulfur Radioisotopes; C058380:naloxone benzoylhydrazone; D006153:Guanosine Diphosphate; D009270:Naloxone; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004441:Ecdysterone; C008999:ponasterone A; D000094942:Nociceptin Receptor