Juxtaposition of the Steroid Binding Domain-like I and II Regions Constitutes a Ligand Binding Site in the σ-1 Receptor

Arindam Pal,Uyen B Chu,Subramaniam Ramachandran,David Grawoig,Lian-Wang Guo,Abdol R Hajipour,Arnold E Ruoho

doi:10.1074/jbc.m802192200

Abstract

sigma-1 receptors represent unique binding sites that are capable of interacting with a wide range of compounds to mediate different cellular events. The composition of the ligand binding site of this receptor is unclear, since no NMR or crystal structures are available. Recent studies in our laboratory using radiolabeled photoreactive ligands suggested that the steroid binding domain-like I (SBDLI) (amino acids 91-109) and the steroid binding domain-like II (SBDLII) (amino acids 176-194) regions are involved in forming the ligand binding site(s) ( Chen, Y., Hajipour, A. R., Sievert, M. K., Arbabian, M., and Ruoho, A. E. (2007) Biochemistry 46, 3532-3542 ; Pal, A., Hajipour, A. R., Fontanilla, D., Ramachandran, S., Chu, U. B., Mavlyutov, T., and Ruoho, A. E. (2007) Mol. Pharmacol. 72, 921-933 ). In this report, we have further addressed this issue by utilizing our previously developed sulfhydryl-reactive, cleavable, radioiodinated photocross-linking reagent: methanesulfonothioic acid, S-((4-(4-amino-3-[125I]iodobenzoyl) phenyl)methyl) ester (Guo, L. W., Hajipour, A. R., Gavala, M. L., Arbabian, M., Martemyanov, K. A., Arshavsky, V. Y., and Ruoho, A. E. (2005) Bioconjugate Chem. 16, 685-693). This photoprobe was shown to derivatize the single cysteine residues as mixed disulfides at position 94 in the SBDLI region of the wild type guinea pig sigma-1 receptor (Cys94) and at position 190 in the SBDLII region of a mutant guinea pig sigma-1 receptor (C94A,V190C), both in a sigma-ligand (haloperidol or (+)-pentazocine)-sensitive manner. Significantly, photocross-linking followed by Endo Lys-C cleavage under reducing conditions and intramolecular radiolabel transfer from the SBDLI to the SBDLII region in the wild type receptor and, conversely, from the SBDLII to the SBDLI region in the mutant receptor were observed. These data support a model in which the SBDLI and SBDLII regions are juxtaposed to form, at least in part, a ligand binding site of the sigma-1 receptor.

Highlights

The ␴ receptors represent unique nonopioid and nonphencyclidine binding sites that are distinct from other known neurotransmitters or hormone receptors [4]
The cloned ␴-1 receptor has 223 amino acids and shares 30% identity and 67% similarity with a yeast sterol C8-C7 isomerase (ERG2), which is involved in cholesterol synthesis [43]
Juxtaposition of SBDLI/SBDLII Regions in ␴-1 Receptor segment was proposed as a transmembrane domain (TMD)2 [37], and the other two hydrophobic segments, were proposed previously as the steroid binding domain-like I (SBDLI) and steroid binding domain-like II (SBDLII) regions due to high sequence homology with the steroid binding domain of the ERG2 [1]

Summary

Introduction

The ␴ receptors represent unique nonopioid and nonphencyclidine binding sites that are distinct from other known neurotransmitters or hormone receptors [4]. This report supports our previously proposed model [2] in which the SBDLI and SBDLII regions are juxtaposed to form, in part, a ligand binding site of the ␴-1 receptor.

Results

Conclusion