Identification of regions of the σ-1 receptor ligand binding site using a novel photoprobe

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-azido3-[ I-125] iodo-phenyl) propane ([I-125] 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 [I-125] 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 [I-125]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 [I-125]IAF interaction with the sigma-1 receptor was identified by (+)-[H-3] pentazocine competitive binding with nonradioactive [I-127] 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).