Vol. 286, Issue 2, 896-902, August 1998

Nociceptin (Orphanin FQ): High-Affinity and High-Capacity Binding Site Coupled to Low-Potency Stimulation of Guanylyl-5'-O-(-thio)-triphosphate Binding in Rat Brain Membranes¹

Erika Albrecht, Nelya N. Samovilova, Stephan Oswald, Ingo Baeger and Hartmut Berger

Department of Peptide Pharmacology (E.A., S.O., I.B., H.B.), Research Institute of Molecular Pharmacology, Berlin, Germany and Cardiology Research Centre (N.N.S.), Academy of Medical Sciences, Moscow, Russia

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-(-thio)-triphosphate (GTPS). Using ³H-Tyr¹⁴- and ¹²⁵I-Tyr¹⁴-nociceptin in saturation and displacement receptor binding studies, a single high-affinity (K_d 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 of ³⁵S-GTPS 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 K_d 45.8 to 1.57 nM of only about 6.5% of the basal binding sites for GTPS, 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 of ³⁵S-GTPS 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 GTPS 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.