The little understood sigma-1 receptor was originally thought to be a member of the opioid/cannabinoid receptor family. However, it is also overexpressed in many types of human cancer while none was found in samples from neighboring normal tissue. It was deduced to play a role in sustaining survival by providing a powerful anti-apoptotic signal during proliferation and by preventing anoikis during release of cells from the primary tumor and invasion of normal physiological boundaries. The sigma-1 receptor has been utilized in radioimmunoscintigraphy and has recently been suggested as a target for therapy in cancer. The sigma-1 receptor contains two putative transmembrane domains, one at the N-terminus and one in the center of the sequence. Attempts to express and purify the complete sequence from bacterial cells for structural studies using different vectors and host strains yielded very little full-length recombinant protein, and none of it was in a soluble form. Studies with deletion mutants showed that removal of the N-terminal transmembrane domain greatly increased the yield although the majority of the expressed protein remained insoluble. The insoluble protein is being solubilized, refolded and purified. Using RT-PCR and western blotting, we showed that sigma-1 mRNA and protein were present in 26 of a panel of 30 human carcinoma cell lines derived from different tissues. All the breast, renal, vulval, pancreatic, lung, melanoma, ovarian and 75% of colon cancers cell lines tested expressed the highest level of sigma-1 receptors whereas 83% of head and neck squamous carcinoma cell lines expressed moderate levels. In contrast, only 50% of the prostate cancer cell lines had detectable sigma-1 mRNA and protein. In order to follow the cellular dynamics, we have also expressed a fluorescently-tagged full length sigma-1 receptor protein, which has GFP at the C-terminus, in several human tumor cell lines with differentially activated signalling pathways. Colocalization studies using confocal microscopy revealed that the sigma-1 fusion protein was extra-nuclear, and appeared to be concentrated inside cellular organelles, most likely on the ER membrane. Immunostaining showed a similar distribution of native sigma-1 within the cells showing that the fluorescence tag is not affecting the localisation of the receptor. Studies using differential centrifugation are underway to clarify its precise subcellular distribution and to determine whether it translocates following ligand binding. Interactions of the fluorescent sigma-1 fusion protein with potential binding partners and effects of agonistic and antagonistic ligands are being investigated using FRET and FCCS to uncover the major signalling pathways of sigma-1 receptor in cancer cells.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]