Photoaffinity labeling of the Sigma-1 Receptor with N-(3-(4-nitrophenyl)propyl)-N-dodecylamine (4-NPPC12) – Evidence for Receptor Dimers

摘要

The sigma-1 receptor is a ligand-regulated ER resident chaperone involved in the maintenance of cellular homeostasis. Coupling of the sigma-1 receptor with various ER and/or plasma membrane ion channels is associated with its ability to regulate the locomotor activity and cellular proliferation produced in response to sigma-1 receptor ligands. A number of endogenous small molecules bind to the sigma-1 receptor and have been shown to regulate its activity; these include progesterone, N,N-dimethyltryptamine, D-erythro-sphingosine, and/or other endogenous lipids. We previously reported the synthesis of long chain N-alkylamine derivatives and the characterization of the structural-activity relationship between chain length of N-alkylamine and affinities at the sigma-1 receptor. Here, we present data demonstrating the photo-incorporation of one of these N-alkylamine derivatives, 4-NPPC12, to the sigma-1 receptor. MALDI-TOF-TOF and tandem mass spectrometry showed that 4-NPPC12 photo-inserted at histidine 154 of the derivatized population of the sigma-1 receptor. Interestingly, light dependent photo-insertion of 4-NPPC12 resulted in an enhanced electrophoretic mobility of only 50% of the derivatized receptor molecules as assessed by SDS polyacrylamide gel electrophoresis. The proposed binding and reactivity of 4-NPPC12 evokes a ligand binding model for the sigma-1 receptor that likely involves a receptor dimer and/or oligomer.