Abstract

The sigma 1 receptor ({sigma}1R) has been implicated in cancers, neurological disorders, and substance use disorders. Yet, its molecular and cellular functions have not been well-understood. Recent crystal structures of {sigma}1R reveal a single N-terminal transmembrane segment and C-terminal ligand-binding domain, and a trimeric organization. Nevertheless, outstanding issues surrounding the functional or pharmacological relevance of {sigma}1R oligomerization remain, such as the minimal protomeric unit and the differentially altered oligomerization states by different classes of ligands. Western blot (WB) assays have been widely used to investigate protein oligomerizations. However, the unique topology of {sigma}1R renders several intertwined challenges in WB. Here we describe a WB protocol without temperature denaturization to study the ligand binding effects on the oligomerization state of {sigma}1R. Using this approach, we observed unexpected ladder-like incremental migration pattern of {sigma}1R, demonstrating preserved homomeric interactions in the detergent environment. We compared the migration patterns of intact {sigma}1R construct and the C-terminally tagged {sigma}1R constructs, and found similar trends in response to drug treatments. In contrast, N-terminally tagged {sigma}1R constructs show opposite trends to that of the intact construct, suggesting distorted elicitation of the ligand binding effects on oligomerization. Together, our findings indicate that the N-terminus plays an important role in eliciting the impacts of bound ligands, whereas the C-terminus is amenable for modifications for biochemical studies.