Abstract
G protein activation by the agonist-occupied nociceptin- (orphanin FQ-) receptor in rat cerebral cortex was studied by characterizing the nociceptin-stimulated binding of the radiolabeled guanylyl triphosphate (GTP) analog 35S-guanylyl-5′-O-(γ-thio)-triphosphate (GTPγS). Using 3H-Tyr14- and125I-Tyr14-nociceptin in saturation and displacement receptor binding studies, a single high-affinity (Kd 21.6–116.7 pM) and high-capacity binding site for nociceptin (orphanin FQ) in membranes and sections of rat cerebral cortex was identified. Stable GTP analogs and NaCl lowered the affinity only moderately by 2- to 3-fold, but under these conditions nociceptin stimulated the binding of35S-GTPγS to G proteins in the membranes with a potency about 100-fold lower (EC50 9.11 nM). It was estimated that this stimulation was due to a 29-fold increase in the affinity from Kd 45.8 to 1.57 nM of only about 6.5% of the basal binding sites for GTPγS, and that at least 10 G protein binding sites could be stimulated by one receptor site. The link of this nociceptin-stimulated binding of GTP to the nociceptin receptor was further evidenced by the specificity of stimulation, as seen with nociceptin, nociceptin(1–13),d-Ala7-nociceptin and nociceptin(1–9), which paralleled that of their receptor affinities. Furthermore, the distribution in rat brain regions of the binding of35S-GTPγS stimulated by nociceptin differed from that stimulated by the mu opioid agonist [d-Ala2, N-Me-Phe4, Gly5-ol)]-enkephalin. Especially, no stimulation by nociceptin was observed in caudate putamen, where also the absence of ORL1 receptors had been reported. The putative coupling of the high-affinity nociceptin receptor to the low-potency stimulation of GTPγS binding in rat cerebral cortex might be explained by the switch of a low part of occupied nociceptin binding sites to a very low-affinity state being stabilized at high peptide concentrations and catalytically stimulating the GTP binding.
Footnotes
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Send reprint requests to: Dr. Hartmut Berger, Research Institute of Molecular Pharmacology, Alfred-Kowalke-Str. 4, D-10315 Berlin, F.R.G.
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↵1 This work was supported in part by a grant to N.N.S. from the Deutscher Akademischer Austauschdienst e.V. (Bonn, F.R.G.).
- Abbreviations:
- BSA
- bovine serum albumin
- CHO
- Chinese hamster ovary
- EGTA
- ethylene glycol bis(2-aminoethylether)-N, N,N′,N′-tetraacetic acid
- DAMGO
- [d-Ala2, N-Me-Phe4, Gly5-ol)]-enkephalin
- Gpp(NH)p
- 5′-guanylylimidodiphosphate
- GDP
- guanosine-5′-diphosphate
- GTPγS
- guanylyl-5′-O-(γ-thio)-triphosphate
- HEK
- human embryonic kidney
- ORL1 receptor
- opioid receptor-like receptor 1
- Received November 20, 1997.
- Accepted April 6, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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