Probing the Steroid Binding Domain-like I (SBDLI) of the Sigma-1 Receptor Binding Site Using N-Substituted Photoaffinity Labels

Radioiodinated photoactivatable photoprobes can provide valuable insights regarding protein structure. Previous work in our laboratory showed that the cocaine derivative and photoprobe 3-[125I]iodo-4-azidococaine ([125I]IACoc) binds to the sigma-1 receptor with 2−3 orders of magnitude higher affinity than cocaine [Kahoun, J. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393−1397]. Using this photoprobe, we demonstrated the insertion site for [125I]IACoc to be Asp188 [Chen, Y. (2007) Biochemistry 46, 3532−3542], which resides in the proposed steroid binding domain-like II (SBDLII) region (amino acids 176−194) [Pal, A. (2007) Mol. Pharmacol. 72, 921−933]. An additional photoprobe based on the sigma-1 receptor ligand fenpropimorph, 1-N-(2-3-[125I]iodophenyl)propane ([125I]IAF), was found to label a peptide in both the SBDLII and steroid binding domain-like I (SBDLI) (amino acids 91−109) [Pal, A. (2007) Mol. Pharmacol. 72, 921−933]. In this report, we describe two novel strategically positioned carrier-free, radioiodinated photoaffinity labels specifically designed to probe the putative “nitrogen interacting region” of sigma-1 receptor ligands. These two novel photoprobes are (−)-methyl 3-(benzoyloxy)-8-2-(4-azido-3-[125I]iodobenzene)-1-ethyl-8-azabicyclo[3.2.1]octane-2-carboxylate ([125I]-N-IACoc) and N-propyl-N-(4-azido-3-iodophenylethyl)-3-(4-fluorophenyl)propylamine ([125I]IAC44). In addition to reporting their binding affinities to the sigma-1 and sigma-2 receptors, we show that both photoaffinity labels specifically and covalently derivatized the pure guinea pig sigma-1 receptor (26.1 kDa) [Ramachandran, S. (2007) Protein Expression Purif. 51, 283−292]. Cleavage of the photolabeled sigma-1 receptor using Endo Lys C and cyanogen bromide (CNBr) revealed that the [125I]-N-IACoc label was located primarily in the N-terminus and SBDLI-containing peptides of the sigma-1 receptor, while [125I]IAC44 was found in peptide fragments consistent with labeling of both SBDLI and SBDLII.