Photo‐crosslinking Reveals Unique Features of the Sigma‐1 Receptor Ligand Binding Region(s)

We have recently found that endogenous sphingolipids including sphingosine and sphinganine interact with the sigma‐1 receptor. To study the interaction of these lipid molecules with the receptor, we used a series N ‐alkylamines and their phenylpropyl‐ as well as nitrophenylpropyl‐ derivatives as surrogates for sphingosine. Surprisingly, N ‐(3‐(4‐nitrophenyl)propyl)‐N‐alkylamines, revealed a unique property of the sigma‐1 receptor whereby upon UV irradiation of the receptor in the presence of excess compounds, two sigma‐1 receptor populations were generated. The first population migrated as a 26 kDa and the second as 23 kDa on an SDS polyacrylamide gel with a ratio of 1:1. Using anti‐sigma‐1 receptor antibody, we also detected this phenomenon in both guinea pig liver membranes and COS‐7 cells overexpressing the sigma‐1 receptor. Generation of these two sigma‐1 receptor populations was dependent on photolysis of N ‐alkylamine derivatives of 12 carbons or longer and was protectable by haloperidol. The 23 kDa population was not formed as a result of photo‐chemical cleavage of the sigma‐1 receptor since both of the N‐ and C‐termini are presence as confirmed by N‐terminal sequencing and western analysis using an anti‐C‐term hexahistidine antibody. Sequence specific antibodies against amino acids 143 – 169 of the sigma‐1 receptor showed that in the 23 kDa, the N‐alkylamine photoprobe blocked the antibody epitope of this region. Additionallly, MALDI‐TOF results showed that there is a 318 Da shift of the peptide corresponding to amino acids 143 – 175, suggesting a covalent cross‐linking of photoprobe to the pure protein. Together, these data reveal unique features of the sigma‐1 receptor binding region(s).