Pharmacological profiling of sigma 1 receptor ligands by novel receptor homomer assays.

The sigma 1 receptor (σR) is a structurally unique transmembrane protein that functions as a molecular chaperone in the endoplasmic reticulum (ER), and has been implicated in cancer, neuropathic pain, and psychostimulant abuse. Despite physiological and pharmacological significance, mechanistic underpinnings of structure-function relationships of σR are poorly understood, and molecular interactions of selective ligands with σR have not been elucidated. The recent crystallographic determination of σR as a homo-trimer provides the foundation for mechanistic elucidation at the molecular level. Here we report novel bioluminescence resonance energy transfer (BRET) assays that enable analyses of ligand-induced multimerization of σR and its interaction with BiP. Haloperidol, PD144418, and 4-PPBP enhanced σR homomer BRET signals in a dose dependent manner, suggesting their significant effects in stabilizing σR multimerization, whereas (+)-pentazocine and several other ligands do not. In non-denaturing gels, (+)-pentazocine significantly decreased whereas haloperidol increased the fraction of σR multimers, consistent with the results from the homomer BRET assay. Further, BRET assays examining heteromeric σR-BiP interaction revealed that (+)-pentazocine and haloperidol induced opposite trends of signals. From molecular modeling and simulations of σR in complex with the tested ligands, we identified initial clues that may lead to the differed responses of σR upon binding of structurally diverse ligands. By combining multiple in vitro pharmacological and in silico molecular biophysical methods, we propose a novel integrative approach to analyze σR-ligand binding and its impact on interaction of σR with client proteins.