Binding of GTPγ[35S] is regulated by GDP and receptor activation. Studies with the nociceptin/orphanin FQ receptor

Abstract

Background and purpose: We have examined the effects of ligand efficacy and receptor density on the binding of guanosine 5′‐[γ‐thio]triphosphate (GTPγS) and GDP to the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP)‐coupled G‐proteins.Experimental approach: In GTPγ[35S] binding experiments, using stable (CHOhNOP) and inducible (CHOINDhNOP) 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 GTPγS association kinetics.Key results: GTPγS 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 µM 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)NH2 ([Phe1ψ(CH2‐NH)Gly2]‐nociceptin(1‐13)NH2) and naloxone benzoylhydrazone both reduced the fraction of high‐affinity sites for GDP (relative to basal). While the pIC50 for high‐affinity GDP binding site did not decrease in the presence of 1 µM N/OFQ, N/OFQ produced a significant reduction in pIC50 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 GTPγ[35S] was regulated by GDP and receptor activation caused increased binding of GTPγ[35S] through a reduction in GDP affinity.