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

The σ-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. The authors have developed a synthetic, highly selective ligand for this receptor, 1,3-di-o-tolylguanidine (DTG). To identify the binding protein(s) of the σ receptor, they have now synthesized a radiolabeled azide derivative of DTG, ([3H]N3DTG). In guinea pig brain membrane binding assays conducted in the dark, [3H]N3DTG bound reversibly, selectively, and with high affinity to σ 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 σ 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/sub r/, 150,000) that may represent the intact σ receptor complex. NaDodSO4/PAGE of this complex showed the radiolabeled material was a 29-kDa polypeptide that may be binding subunit of the σ receptor