Abstract
σ Receptors, once considered a class of opioid receptors, are now regarded as a unique class of receptors that contain binding sites for a wide range of ligands, including the drug 1-N(2′,6′-dimethylmorpholino)3-(4-t-butylpropylamine) (fenpropimorph), a yeast sterol isomerase inhibitor. Because fenpropimorph has high-binding affinity to the σ-1 receptor, we have synthesized a series of fenpropimorph-like derivatives with varying phenyl ring substituents and have characterized their binding affinities to the σ-1 receptor. In addition, we have synthesized a carrier-free, radioiodinated fenpropimorph-like photoaffinity label, 1-N-(2′,6′-dimethyl-morpholino)-3-(4-azido-3-[125I]iodo-phenyl)propane ([125I]IAF), which covalently derivatized the σ-1 receptor (25.3 kDa) in both the rat liver and guinea pig liver membranes and the σ-2 receptor (18 kDa) in rat liver membranes with high specificity. Furthermore, after cleaving the specific [125I]IAF-photolabeled σ-1 receptor in guinea pig and rat liver membranes and the pure guinea pig σ-1 receptor with EndoLys-C and cyanogen bromide, the [125I]IAF label was identified both in a peptide containing steroid binding domain-like I (SBDLI) (amino acids 91–109) and in a peptide containing steroid binding domain-like II (SBDLII) (amino acids 176–194). Because a single population of binding sites (R2 = 0.992) for [125I]IAF interaction with the σ-1 receptor was identified by (+)-[3H]pentazocine competitive binding with nonradioactive [127I]IAF, it was concluded that SBDLI (amino acids 91–109) and SBDLII (amino acids 176–194) comprises, at least in part, regions of the σ-1 receptor ligand binding site(s).
Footnotes
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This work was supported by the National Institutes of Health grant R01-MH065503 (to A.E.R.).
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.038307.
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ABBREVIATIONS: SKF-10047, N-allyl-normetazocine; IACoc, 3-iodo-4-azido cocaine; IAF, 1-N-(2′,6′-dimethyl-morpholino)-3-(4-azido-3-iodophenyl) propane; SBDLI, sterol binding domain-like I; SBDLII, sterol binding domain-like II; TMD, transmembrane domain; CNBr, cyanogen bromide; PAGE, polyacrylamide gel electrophoresis; DTG, ditolylguanidine; MBP, maltose binding protein; THF, tetrahydrofuran; TLC, thin-layer chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
- Received May 18, 2007.
- Accepted July 10, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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