title: Identification of the binding subunit of the sigma-type opiate receptor by photoaffinity labeling with 1-(4-azido-2-methyl[6-3H]phenyl)-3-(2-methyl[4,6-3H]phenyl)guanidine.
abstract: The sigma-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 sigma 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 (Kd = 10 nM) to sigma 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 sigma 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 (Mr, 150,000) that may represent the intact sigma 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 sigma receptor. The specific sigma receptor photoaffinity ligand described here should be a useful tool for purifying and characterizing the sigma receptor.
authors.affiliation: Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
authors.forename: M P
authors.initials: MP
authors.lastname: Kavanaugh
authors.forename: B C
authors.initials: BC
authors.lastname: Tester
authors.forename: M W
authors.initials: MW
authors.lastname: Scherz
authors.forename: J F
authors.initials: JF
authors.lastname: Keana
authors.forename: E
authors.initials: E
authors.lastname: Weber
mesh_terms: D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D006146:Guanidines / Q000378:metabolism*; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D010777:Photochemistry; D011957:Receptors, Opioid / Q000378:metabolism*; D017480:Receptors, sigma
chemical_list: D000345:Affinity Labels; D001386:Azides; D006146:Guanidines; D011957:Receptors, Opioid; D017480:Receptors, sigma; C055550:1-(4-azido-2-methylphenyl)-3-(2-methylphenyl)guanidine