Identification of the binding subunit of the δ-type opiate receptor by photoaffinity labeling with 1-(4-azido-2-methyl[6-³H]phenyl)-3-(2-methyl[4,6-³H]phenyl) guanidine

The δ-type opiate receptor is a distinct binding site in the brain that may mediate some of the psychotomimetic effects caused by benzomorphan opiates and phencyclidine in humans. We have developed a synthetic, highly selective ligand for this receptor, 1,3-di-o-tolylguanidine (DTG). To identify the binding protein(s) of the δ receptor, we have now synthesized a radiolabeled azide derivative of DTG, 1-(4-azido-2-methyl[6-³H]phenyl)-3-(2-methyl[4,6-³H]phenyl) guanidine ([³H]N₃DTG). In guinea pig brain membrane binding assays conducted in the dark, [³H]N₃DTG bound reversibly, selectively, and with high affinity K(d) = 10 nM) to δ receptors. The drug specificity profile of reversible [³H]N₃DTG binding was identical to that of [³H]DTG and ³H-labeled (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding indicating that [³H]N₃DTG is a selective δ receptor ligand. Guinea pig brain membranes were photoaffinity-labeled with [³H]N₃DTG. NaDodSO₄/PAGE of detergent-solubilized membrane extract identified a single 29-kDa radioactive band. Sepharose Cl-6B gel chromatography of photolabeled brain membranes solubilized with the nondenaturing detergent sodium cholate showed a radioactive complex with a Stoke's radius of 4.6 nm (M(r), 150,000) that may represent the intact δ receptor complex. NaDodSO₄/PAGE of this complex showed that the radiolabeled material was a 29-kDa polypeptide that may be the binding subunit of the δ receptor. The specific δ receptor photoaffinity ligand described here should be a useful tool for purifying and characterizing the δ receptor.