Distinct Regulation of σ1 Receptor Multimerization by Its Agonists and Antagonists in Transfected Cells and Rat Liver Membranes

Extensive studies have shown that the sigma(1) receptor (sigma R-1) interacts with and modulates the activity of multiple proteins with important biological functions. Recent crystal structures of sigma R-1 as a homotrimer differ from a dimer-tetramer model postulated earlier. It remains inconclusive whether ligand binding regulates sigma R-1 oligomerization. Here, novel nondenaturing gel methods and mutational analysis were used to examine sigma R-1 oligomerization. In transfected cells, sigma R-1 exhibited as multimers, dimers, and monomers. Overall, sigma R-1 agonists decreased, whereas sigma R-1 antagonists increased sigma R-1 multimers, suggesting that agonists and antagonists differentially affect the stability of sigma R-1 multimers. Endogenous sigma R-1 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 sigma R-1 to apparent monomer. These results demonstrate that multiple domains play crucial roles in coordinating high-order quaternary organization of sigma R-1. The E102Q sigma R-1 mutant implicated in juvenile amyotrophic lateral sclerosis formed dimers only, suggesting that dysregulation of sigma R-1 multimeric assembly may impair its function. Interestingly, oligomerization of sigma R-1 was pH-dependent and correlated with changes in [H-3](+)-pentazocine binding affinity and Bmax. Combined with mutational analysis, it is reasoned that sigma R-1 multimers possess high-affinity and high-capacity [H-3](+)-pentazocine binding, whereas monomers likely lack binding. These results suggest that sigma R-1 may exist in interconvertible oligomeric states in a dynamic equilibrium. Further exploration of ligand- regulated sigma R-1 multimerization may provide novel approaches to modulate the function of sigma R-1 and its interacting proteins. SIGNIFICANCE STATEMENT The sigma(1) receptor (sigma R-1) modulates the activities of various partner proteins. Recently, crystal structures of sigma R-1 were elucidated as homotrimers. This study used novel nondenaturing gel methods to examine sigma R-1 oligomerization in transfected cells and rat liver membranes. Overall, agonist binding decreased, whereas antagonist binding increased sigma R-1 multimers, which comprised trimers and larger units. sigma R-1 multimers were shown to bind [H-3](+)-pentazocine with high affinity and high capacity. Furthermore, mutational analysis revealed a crucial role of its N-terminal domain in sigma R-1 multimerization.