The C terminal sequence is crucial for maintaining the struc

The C-terminal sequence 209–223 is crucial for maintaining the structure of the active site, with its deletions leading to loss of the ligand-binding properties [49]. The carboxyl terminus of the first transmembrane domain TM1 is also involved in the formation of the binding site through close interaction with the SBDLI and SBDLII regions located within a distance of 8Å from each other [46,50]. Thus, the hypothetical spatial organization of the ligand-binding site of the sigma-1 receptor is based on the intramolecular juxtaposition of the TM1/SBDLI segments with the SBDLII region. A recent series of papers described building a model of the sigma-1 receptor and mapping its active site by in silico methods and site-directed mutagenesis [51,52]. The initial model was constructed by homology modeling (PDB 3CIA, 1I24, 2Z2Z, 2Q8I), while its non-homologous part (the N-terminal segment) was constructed de novo. The computer simulation results are in good agreement with the currently available experimental data, and the model itself has been used to design and develop new, more specific synthetic ligands of the sigma-1 receptor [53,54]. Mapping of the active site allowed to identify the key residues involved in pentazocine binding, a sigma-1 receptor agonist. Aspartic purchase AVE 0991 residue D126 forms a crucial connection, a salt bridge to the nitrogen atom of the pentazocine molecule; E172 forms a hydrogen bond with the hydroxyl group of the ligand; the R119, I128, Y173 residues form a hydrophobic pocket of the active site. Residue mutations in the SBDLII region had little effect on the binding of ligands, but the C-terminal segment of residues 200–223 turned out to be necessary for stabilizing the structure of the binding site. This model is in good agreement with the hypothesis about the presence of the binding site involving all of the receptor domains. There is evidence that the sigma-1 receptor is functionally active and capable of binding ligands only in oligomeric state [55,56]. The first macromolecular forms of the sigma-1 receptor (molecular masses of 97, 130 and 147kDa) were detected in the microsomal membranes of the rat liver using radioiodinated photoaffinity labels [46]. Further evidence of the existence of the sigma-1 receptor in various forms (both monomeric and oligomeric) was also obtained using spectral FRET imaging in living COS-7 cells [57]. In vitro experiments with the purified recombinant MBP-S1R fusion protein showed that only oligomeric forms of the sigma-1 receptor (hexamers, tetramers, and octamers) are capable of binding the tritiated agonist [3H]–(+)-pentazocine, while the monomers are functionally inactive [55]. Haloperidol, which is a well-known antagonist, promotes high-molecular oligomeric forms, but pentazocine, which is an agonist, stabilizes the dimers of the sigma-1 receptor [57]. The ability to oligomerize is associated with the structural features of the sigma-1 receptor. Two presumed GxxxG dimerization motifs were found in the receptor sequence [56,58,59]. The first of them is located in the TM2 domain (residues 87–91), while the second one is in the C-terminal region of SBDLII (residues 108–112). It is possible that the first motif mediates the dimerization of the sigma-1 receptor, and the other is responsible for the formation of high molecular weight oligomers. All point mutations in the oligomerization domain (GGWMG, residues 87–91) led to a significant decrease in the expression of the receptor in cells and also to a shift of the oligomeric forms of the receptor toward monomers [55]. The truncated form of the sigma receptor-1 (residues 1–122) is capable of forming heteromers with the full-length version of the receptor, negatively regulating the function of the full-length version [42]. Chu et al. proposed a model in which the sigma-1 receptor forms a homodimer or an oligomer containing dimers, with the 1:1 binding stoichiometry of ligand to dimer of the sigma-1 receptor [56]. In contrast with this model, a number of other computer simulation studies demonstrate that it is possible for ligands to bind to a monomeric form of the sigma-1 receptor, which corresponds to the stoichiometry of one ligand per one receptor [51–53].