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 (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 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 K_d 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.