John McDonald1, David G Lambert. 1. Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester Royal Infirmary, Leicester, UK.
Abstract
BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY 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 AND IMPLICATIONS: 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.
BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY 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 AND IMPLICATIONS: 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.
Authors: Justyna Piekielna-Ciesielska; Roberto Artali; Ammar A H Azzam; David G Lambert; Alicja Kluczyk; Luca Gentilucci; Anna Janecka Journal: Molecules Date: 2020-12-22 Impact factor: 4.411