The effects of terminal tagging on homomeric interactions of the sigma 1 receptor

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 Cterminal 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 σ1Rconstruct 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.

The Sigma-1 receptor is an ER-localized type II membrane protein

“…This is likely due to the short N-terminal tail and thus the proximity of the tag to the TMD. N-terminal tagging was also reported to affect S1R oligomerization (32), which we confirmed with BAP-S1R. A study using an ascorbate peroxidase 2 (APEX2) tagging approach indicated a type II orientation, thus in this case the tagging did not seem to affect the orientation of the protein (29).…”

“…We analyzed whether the tags affect the oligomerization of the S1R. For this, we separated Triton-insoluble species (higher order oligomers (31)), and kept the lysates at 4˚C before SDS-PAGE, which prevents dissociation of S1R dimers (32). To avoid the formation of J o u r n a l P r e -p r o o f mixed oligomers, we performed these experiments by expressing the constructs in HEK293 cells where S1R was deleted using CRISPR/Cas9 technology (S1R-/-) (15).…”

Effect of degree of neutralization of sodium‐based ionomers on hot‐tack properties

“…[15] Ionomers are currently used as adhesive materials for packaging because they exhibit superior heat welding properties, meaning the ability to melt and adhere to other substances in response to thermal energy. The characteristics of ionomers include high adhesive strength, [16][17][18] improved low-temperature sealing, [16][17][18] and greater adhesion to dissimilar materials [17] compared with other resins. Heat welding properties can be divided into two categories, associated with heat-sealing and hottack behavior.…”