Received for publication May 22, 2007.
Revised July 10, 2007.
Accepted for publication July 10, 2007.

Identification of Regions of the Sigma-1 Receptor Ligand Binding Site Using a Novel Photoprobe

Abstract

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 fenpropimorph (1-N(2',6'-dimethylmorpholino)3-(4-t-butylpropylamine)), a yeast sterol isomerase inhibitor. Since 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. Additionally, 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 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 [¹²⁵I]IAF photolabeled sigma-1 receptor in guinea pig and rat liver membranes as well as the pure guinea pig sigma-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). Since a single population of binding sites (R² = 0.992) for [¹²⁵I]IAF interaction with the sigma-1 receptor was identified by (+)-[³H]pentazocine competitive binding with non-radioactive [¹²⁷I]IAF, it is concluded that SBDLI (sequence 91-109) and SBDLII (amino acids 176-194) comprise, at least in parts, regions of the sigma-1 receptor ligand binding site(s).

Key words: Opioid, Orphan, Ion channel regulation, Molecular dynamics, Structure determinations, Structure-activity relationships and modeling, Prediction of structure-function/proteomics, Anti-psychotics, Cocaine, Opioids

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