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CNS Research, Pharma Division, F. Hoffmann-La Roche AG, CH-4070
Basel, Switzerland
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 Kd 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
-(
,
-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.
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