Interaction of [³H]Orphanin FQ and¹²⁵I-Tyr14-Orphanin FQ with the Orphanin FQ Receptor: Kinetics and Modulation by Cations and Guanine Nucleotides

Abstract

The heptadecapeptide orphanin FQ (OFQ) has been identified as the endogenous ligand for a G protein-coupled receptor (OFQ-R), which, despite its high degree of sequence similarity to opioid receptors, fails to bind opioid ligands. We developed two radioligands for the OFQ-R: a tritiated native OFQ peptide ([³H]OFQ) and a radioiodinated form in which Leu14 was substituted by tyrosine (¹²⁵I-Tyr14-OFQ). Their binding properties were examined in human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells heterologously expressing the OFQ-R at different levels (HEK 293 expressed 40-fold more OFQ-R than did CHO). Both ligands exhibited rapid, monophasic association kinetics in each cell line. Dissociation of both ligands from OFQ-R expressed in HEK 293 cells was biphasic, whereas dissociation of ¹²⁵I-Tyr14-OFQ from OFQ-R expressed in CHO cells was monophasic and slow. Saturation binding analysis revealed two affinity states in HEK 293 cells with binding parameters in accord with those determined kinetically. In CHO cells,¹²⁵I-Tyr14-OFQ detected a single affinity state with an intermediate K_d value of 54 pm. Optimal binding of the radioligands required 1–5 mm MgCl₂, whereas millimolar concentrations of ZnCl₂, CaCl₂, MnCl₂, and NaCl reduced specific binding of both ligands. A nonhydrolyzable GTP analog [guanosine-5′-(β,γ-imido)triphosphate] reduced the affinity of both OFQ ligands to their receptor without significant changes in the total binding capacity, indicating functional interactions between the OFQ-R and G proteins. In rat brain membranes, specific, saturable binding of ¹²⁵I-Tyr14-OFQ was demonstrated to be pharmacologically identical to the heterologously expressed OFQ-R. Taken together, these results indicate that ¹²⁵I-Tyr14-OFQ and [³H]OFQ exhibit virtually identical characteristics and are suitable for the pharmacological analysis of the OFQ-R.