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

作者：McDonald John; Lambert David G^*

来源：British Journal of Pharmacology, 2010, 159(6): 1286-1293.

DOI：10.1111/j.1476-5381.2009.00621.x

• nociceptin
• orphanin FQ
• opioids
• guanine nucleotides
• binding

Background and purpose:
We have examined the effects of ligand efficacy and receptor density on the binding of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) and GDP to the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP)-coupled G-proteins.
Experimental approach:
In GTP gamma[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 GTP gamma S association kinetics.
Key results:
GTP gamma S 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 mu M 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 ([Phe1 Sigma(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 pIC(50) for high-affinity GDP binding site did not decrease in the presence of 1 mu M 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 GTP gamma[35S] was regulated by GDP and receptor activation caused increased binding of GTP gamma[35S] through a reduction in GDP affinity.

• 出版日期2010-3