INTERACTION OF [H-3] ORPHANIN FQ AND I-125 TYR14-ORPHANIN FQ WITH THEORPHANIN FQ RECEPTOR - KINETICS AND MODULATION BY CATIONS AND GUANINE-NUCLEOTIDES

The heptadecapeptide orphanin FQ (OFQ) has been identified as the endo genous 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 tr itiated native OFQ peptide ([H-3]OFQ) and a radioiodinated form in whi ch Leu14 was substituted by tyrosine (I-125-Tyr14-OFQ). Their binding properties were examined in human embryonic kidney (HEK) 293 and Chine se hamster ovary (CHO) cells heterologously expressing the OFQ-R at di fferent levels (HEK 293 expressed 40-fold more OFQ-R than did CHO). Bo th ligands exhibited rapid, monophasic association kinetics in each ce ll line. Dissociation of both ligands from OFQ-R expressed in HEK 293 cells was biphasic, whereas dissociation of I-125-Tyr14-OFQ from OFQ-R expressed in CHO cells was monophasic and slow. Saturation binding an alysis revealed two affinity states in HEK 293 cells with binding para meters in accord with those determined kinetically. In CHO cells, I-12 5-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 Mg Cl2, whereas millimolar concentrations of ZnCl2, CaCl2, MnCl2, and NaC l reduced specific binding of both ligands. A nonhydrolyzable GTP anal og [guanosine-5'-(beta,gamma-imido)triphosphate] reduced the affinity of both OFQ ligands to their receptor without significant changes in t he total binding capacity, indicating functional interactions between the OFQ-R and G proteins. In rat brain membranes, specific, saturable binding of I-125-Tyr14-OFQ was demonstrated to be pharmacologically id entical to the heterologously expressed OFQ-R. Taken together, these r esults indicate that I-125-Tyr14-OFQ and [H-3]OFQ exhibit virtually id entical characteristics and are suitable for the pharmacological analy sis of the OFQ-R.