Abstract
Extensive studies have shown that the sigma-1 receptor (σ1R) interacts with and modulates the activity of multiple proteins with important biological functions. Recent crystal structures of σ1R as a homo-trimer differ from a dimer-tetramer model postulated earlier. It remains inconclusive whether ligand binding regulates σ1R oligomerization. Here novel non-denaturing gel methods and mutational analysis were used to examine σ1R oligomerization. In transfected cells σ1R exhibited as multimers, dimers and monomers. Overall σ1R agonists decreased, whereas σ1R antagonists increased σ1R multimers, suggesting that agonists and antagonists differentially affect the stability of σ1R multimers. Endogenous σ1R in rat liver membranes also showed similar regulation of oligomerization as in cells. Mutations at key residues lining the trimerization interface (Arg119, Asp195, Phe191, Trp136, and Gly91) abolished multimerization without disrupting dimerization. Intriguingly, truncation of the N-terminus reduced σ1R to apparent monomer. These results demonstrate that multiple domains play crucial roles in coordinating high-order quaternary organization of σ1R. The E102Q σ1R mutant implicated in juvenile amyotrophic lateral sclerosis formed dimers only, suggesting that dysregulation of σ1R multimeric assembly may impair its function. Interestingly, oligomerization of σ1R was pH dependent and correlated with changes in [3H](+)-pentazocine binding affinity and Bmax. Combined with mutational analysis, it is reasoned that σ1R multimers possess high-affinity and high-capacity [3H](+)-pentazocine binding, whereas monomers likely lack binding. These results suggest that σ1R may exist in interconvertible oligomeric states in a dynamic equilibrium. Further exploration of ligand-regulated σ1R multimerization may provide novel approaches to modulate the function of σ1R and its interacting proteins.
SIGNIFICANCE STATEMENT The σ1R modulates the activities of various partner proteins. Recently crystal structures of σ1R were elucidated as homotrimers. This study used novel non-denaturing gel methods to examine σ1R oligomerization in transfected cells and rat liver membranes. Overall agonist binding decreased whereas antagonist binding increased σ1R multimers, which comprised trimers and larger units. σ1R multimers were shown to bind [3H](+)-pentazocine with high-affinity and high-capacity. Further, mutational analysis revealed a crucial role of its N-terminal domain in σ1R multimerization.
- The American Society for Pharmacology and Experimental Therapeutics