Featured Article - September 2014
Short description: Stabilization of oligomers by small-molecule ligands suggests a mechanism for regulating a membrane protein chaperone.
Mammalian protein sigma 1 receptor (S1R) is a membrane-bound protein that functions as a molecular chaperone for a variety of receptors and voltage-gated ion channels. S1R is implicated in drug addiction and neurodegenerative diseases, and is regulated by a broad range of endogenous ligands such as sphingosine and steroids, as well as synthetic and naturally occurring small-molecule molecules, including cocaine and haloperidol. Previous studies suggested that S1R contains two transmembrane helices and a cytoplasmic C-terminal domain; the latter is involved in chaperone activity, whereas the ligand-binding region is formed by a short hydrophobic portion in the C-terminal domain, a portion of TM2 and possibly a portion of TM1.
To understand the mechanism of S1R activation, Fox and colleagues (PSI TMPC) examined the possibility that S1R oligomerizes under physiological conditions, as suggested by previous results from the Ruoho laboratory. The authors purified protein dodecyl maltoside detergent micelles of guinea pig S1R fused to maltose-binding protein (MBP). S1R eluted as at least two species on analytical size-exclusion chromatography (SEC), both before and after MBP was cleaved away. SEC and light-scattering analyses indicated that the low molecular weight (MW) peak corresponded to a monomer, the high MW peak to oligomeric states of 6–8, and the intermediate MW peak to a tetramer (only seen for MBP-fused S1R). These oligomerization states were supported by SDS-PAGE analysis following treatment with a crosslinker agent. Dissociation of high-MW oligomers into monomers increased with temperature, and incubation with a variety of known S1R ligands reduced heat-induced dissociation. Assaying isolated monomers and oligomers revealed that only the oligomers had ligand-binding activity.
Based on these results, the authors propose that ligand binding may regulate chaperone activity by sequestering S1R into oligomeric states unable to bind client proteins. To determine whether the GxxxG motif in TM2 promotes helix-helix association as seen in other transmembrane proteins, glycine residues 87, 88 and 91 were mutated to isoleucine or leucine. These point mutations drastically reduced the yield of purified S1R protein, markedly shifting the distribution from oligomeric to monomeric forms, and reduced [3H]-(+)-pentazocine binding at the single concentration tested. The latter results are consistent with previous findings by Ruoho and colleagues, implicating residues 91–109 on TM2 in ligand binding.
This work points to possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small-molecule ligands. Further analysis is needed to determine how ligand binding promotes S1R oligomerization and the impact on chaperone activity.
K. Gromek et al. The oligomeric states of the purified sigma 1 receptor are stabilized by ligands.
J Biol Chem. 289, 20333-20344 (2014). doi:10.1074/jbc.M113.537993