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

M. P. Kavanaugh, B. C. Tester, M. W. Scherz, J. F.W. Keana, E. Weber

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)2844-2848
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number8
DOIs
StatePublished - 1988

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