Abstract
The sigma 1 receptor (σ1R) 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 σ1R 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 σ1R 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 σ1R 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 σ1R. Using this approach, we observed unexpected ladder-like incremental migration pattern of σ1R, demonstrating preserved homomeric interactions in the detergent environment. We compared the migration patterns of intact σ1R construct and the C-terminally tagged σ1R constructs, and found similar trends in response to drug treatments. In contrast, N-terminally tagged σ1R 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.
Highlights
The sigma 1 receptor (σ1R) is a structurally unique transmembrane protein found in the endoplasmic reticulum (ER) and is associated with intracellular membranes in which it has been posited to act as a molecular chaperone (Su et al, 2016)
We used an antibody-based western blot (WB) approach to detect the configurational changes of σ1R oligomerization in response to ligand binding and to
In addition to our previous σ1R construct used in the bioluminescence resonance energy transfer (BRET) assay in which Nluc is attached to the C-terminus (σ1R-Nluc) (Yano et al, 2018), to test the C-terminal tagging’s effect on the σ1R-σ1R interaction, we generated the σ1R-myc construct, which has a shorter tag at the C-terminus
Summary
The sigma 1 receptor (σ1R) is a structurally unique transmembrane protein found in the endoplasmic reticulum (ER) and is associated with intracellular membranes in which it has been posited to act as a molecular chaperone (Su et al, 2016). The recent crystal structures of σ1R in complexes with a variety of ligands provide the foundation for mechanistic elucidation of its function at the molecular level (Schmidt et al, 2016, 2018) They shed light on the conformation and homomeric status of Terminal Tagging of the Sigma 1 Receptor σ1R in the membrane environment. Functional categorizations of σ1R ligands remain challenging (Katz et al, 2017) Both the differences in intra- or extracellular environments in various σ1R assays and the lack of functional readouts detecting changes in σ1R itself may have contributed to the difficulty in reaching consensus conclusions. It begs the need for an assay that directly monitors changes occurring in σ1R
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