title: Identification of regions of the sigma-1 receptor ligand binding site using a novel photoprobe.
abstract: sigma 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 sigma-1 receptor, we have synthesized a series of fenpropimorph-like derivatives with varying phenyl ring substituents and have characterized their binding affinities to the sigma-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-[(125)I]iodo-phenyl)propane ([(125)I]IAF), which covalently derivatized the sigma-1 receptor (25.3 kDa) in both the rat liver and guinea pig liver membranes and the sigma-2 receptor (18 kDa) in rat liver membranes with high specificity. Furthermore, after cleaving the specific [(125)I]IAF-photolabeled sigma-1 receptor in guinea pig and rat liver membranes and the pure guinea pig sigma-1 receptor with EndoLys-C and cyanogen bromide, the [(125)I]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 (R(2) = 0.992) for [(125)I]IAF interaction with the sigma-1 receptor was identified by (+)-[(3)H]pentazocine competitive binding with nonradioactive [(127)I]IAF, it was concluded that SBDLI (amino acids 91-109) and SBDLII (amino acids 176-194) comprises, at least in part, regions of the sigma-1 receptor ligand binding site(s).
authors.affiliation: Department of Pharmacology, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706, USA.
authors.forename: Arindam
authors.initials: A
authors.lastname: Pal
authors.forename: Abdol R
authors.initials: AR
authors.lastname: Hajipour
authors.forename: Dominique
authors.initials: D
authors.lastname: Fontanilla
authors.forename: Subramaniam
authors.initials: S
authors.lastname: Ramachandran
authors.forename: Uyen B
authors.initials: UB
authors.lastname: Chu
authors.forename: Timur
authors.initials: T
authors.lastname: Mavlyutov
authors.forename: Arnold E
authors.initials: AE
authors.lastname: Ruoho
mesh_terms: D000818:Animals; D001665:Binding Sites; D006168:Guinea Pigs; D008024:Ligands; D008862:Microsomes, Liver / Q000378:metabolism; D015335:Molecular Probes; D009025:Morpholines / Q000378:metabolism / Q000494:pharmacology; D019906:Nuclear Magnetic Resonance, Biomolecular; D019920:Photoaffinity Labels; D051381:Rats; D017480:Receptors, sigma / Q000378:metabolism*; D000097605:Sigma-1 Receptor
chemical_list: D008024:Ligands; D015335:Molecular Probes; D009025:Morpholines; D019920:Photoaffinity Labels; D017480:Receptors, sigma; D000097605:Sigma-1 Receptor; C050435:fenpropimorph