Nociceptin (orphanin FQ): high-affinity and high-capacity binding site coupled to low-potency stimulation of guanylyl-5'-O-(gamma-thio)-triphosphate binding in rat brain membranes.
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-(gamma-thio)-triphosphate (GTPgammaS). Using 3H-Tyr14- and 125I-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 of 35S-GTPgammaS 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 GTPgammaS, 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-GTPgammaS 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 GTPgammaS 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.
Department of Peptide Pharmacology, Research Institute of Molecular Pharmacology, Berlin, Germany.
E
E
Albrecht
N N
NN
Samovilova
S
S
Oswald
I
I
Baeger
H
H
Berger
D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D001923:Brain Chemistry / Q000187:drug effects*; D002021:Buffers; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D018847:Opioid Peptides / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D011957:Receptors, Opioid / Q000187:drug effects*; D013116:Spinal Cord / Q000187:drug effects / Q000378:metabolism; D000094942:Nociceptin Receptor; D000097629:Nociceptin
D000701:Analgesics, Opioid; D002021:Buffers; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D018847:Opioid Peptides; D011957:Receptors, Opioid; D000094942:Nociceptin Receptor; D000097629:Nociceptin; C000722285:Oprl1 protein, rat