Sigma receptors
Sigma receptors were first classified as opioid receptors, but were later shown to be evolutionary distinct proteins that bind diverse classes of psychotropic drugs. Sigma receptors can be classified into two subtypes, sigma-1 and sigma-2 receptors. Sigma-1 receptor (σ1R) is found in the mammalian central nervous system and most peripheral tissues. It has been suggested that σ1R functions as an endoplasmatic chaperone protein and a regulator of calcium signalling. σ1R is predominantly confined to the endoplasmatic reticulum (ER), specifically to the mitochondria-associated ER, but it also localizes to the nuclear envelope. Upon stimulation σ1R has been shown to translocate to the cell plasma membrane, where it can form oligomers with GPCRs, e.g., dopamine receptors.
We are developing fluorescent markers for the study of translocation of σ1R and its interactions with GPCRs in live cells. σ1R-YFP conjugate is expressed together with different fluorescent markers to detect the translocation of σ1R-YFP in an overexpressed system by co-localization and by possible FRET studies. We have developed Bac-Mam viruses for the expression of σ1R-YFP, mRFP-KDEL, mKate2-mito and WFS1-eGFP conjugates in mammalian cells. mKate2-mito is a conjugate of the fluorescent protein mKate2 and the mitochondrial targeting sequence. WFS1-eGFP is a wolframin ER transmembrane glucoprotein fused to the fluorescent protein eGFP. mRFP-KDEL is a conjugate of the fluorescent protein mRFP and the ER-targeting sequence. In addition to these markers, we have selective fluorescent ligands for dopamine receptor subtypes and stable cell lines expressing dopamine receptors (D1R, D2R and D3R) (Reinart-Okugbeni et al. 2013).
Our studies with the σ1R-YFP conjugate expressed in human ovarian adenocarcinoma cells (SK-OV-3) cells have not confirmed the translocation of σ1R to the cell plasma membrane upon stimulation with a ligand.
We are developing fluorescent markers for the study of translocation of σ1R and its interactions with GPCRs in live cells. σ1R-YFP conjugate is expressed together with different fluorescent markers to detect the translocation of σ1R-YFP in an overexpressed system by co-localization and by possible FRET studies. We have developed Bac-Mam viruses for the expression of σ1R-YFP, mRFP-KDEL, mKate2-mito and WFS1-eGFP conjugates in mammalian cells. mKate2-mito is a conjugate of the fluorescent protein mKate2 and the mitochondrial targeting sequence. WFS1-eGFP is a wolframin ER transmembrane glucoprotein fused to the fluorescent protein eGFP. mRFP-KDEL is a conjugate of the fluorescent protein mRFP and the ER-targeting sequence. In addition to these markers, we have selective fluorescent ligands for dopamine receptor subtypes and stable cell lines expressing dopamine receptors (D1R, D2R and D3R) (Reinart-Okugbeni et al. 2013).
Our studies with the σ1R-YFP conjugate expressed in human ovarian adenocarcinoma cells (SK-OV-3) cells have not confirmed the translocation of σ1R to the cell plasma membrane upon stimulation with a ligand.
Figure 1: A. Expression of σ1R-YFP in SK-OV-3 cells (60x obj.) B. Expression of mKate2-mito in SK-OV-3 cells (60x obj.) C. Expression of mRFP-KDEL in SK-OV-3 cells (20x obj.) D. Binding of the Cy3B-NAPS (the conjugate of the fluorescent dye Cy3B and the D2-type selective antagonist N-(p-aminophenethyl)-spiperone, NAPS) to the cell plasma membrane of HEK-D2R cells (cNAPS-Cy3B = 1 nM, obj. 20x).