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Ligand-dependent localization and intracellular stability of sigma-1 receptors in CHO-K1 cells.

ABSTRACT: BACKGROUND:Sigma-1 receptors are involved in regulation of neuronal activities presumably through regulation of the activity of ion channels. Sigma-1 receptors also play a role in growth and metastasis of cancerous cells. Intracellular distribution of sigma-1 receptors have been linked to sphingolipid-enriched domains. RESULTS:We report that in CHO-K1 cells sigma-1 receptors target to focal adhesion contacts (FAC) where they colocalize with Talin and Kv1.4 potassium channels. The levels of sigma-1 receptors in the FAC were significantly increased by application of sigma-1 receptor ligands and by filamentous actin (F-actin) polymerization with phalloidin. The total length of FAC (measured by the focal adhesion marker, talin) was concomitantly increased in the presence of sigma-1 receptors upon phalloidin treatment. Only sigma-1 receptor ligands, however, resulted in an increase of sigma-1 receptors in the FAC, independent of talin. Additionally, a novel approach was utilized to allow an assessment of the half life of endogenous sigma-1 receptors in CHO-K1 cells, which was measured to be at least 72 hours. CONCLUSION:Ligand activated sigma-1 receptors translocate into FAC from a pool of receptors stored in ER lipid rafts presumably for inhibition of Kv1.4 channels. Stabilization of actin filaments is likely to be important for targeting sigma-1 receptors to Focal Adhesion Contacts in CHO-K1 cells.

SUBMITTER: Mavlyutov TA

PROVIDER: S-EPMC2045653 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Ligand-dependent localization and intracellular stability of sigma-1 receptors in CHO-K1 cells.

Mavlyutov Timur A TA Ruoho Arnold E AE

Journal of molecular signaling 20070920

<h4>Background</h4>Sigma-1 receptors are involved in regulation of neuronal activities presumably through regulation of the activity of ion channels. Sigma-1 receptors also play a role in growth and metastasis of cancerous cells. Intracellular distribution of sigma-1 receptors have been linked to sphingolipid-enriched domains.<h4>Results</h4>We report that in CHO-K1 cells sigma-1 receptors target to focal adhesion contacts (FAC) where they colocalize with Talin and Kv1.4 potassium channels. The ...[more]

PMID: 17883859

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Project description:BACKGROUND: To ensure maximal productivity of recombinant proteins (rP) during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rP. During many such biphasic cultures, the initial phase of rapid cell growth at 37 degrees C is followed by a growth arrest phase induced through reduction of the culture temperature. Low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood. RESULTS: In this study differential protein expression in suspension CHO-K1 cells was investigated following a reduction of the culture temperature from 37 degrees C to 31 degrees C in comparison to standard batch culture maintained at 37 degrees C using 2D-DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry (MS). There is only limited proteomic analysis of suspension-grown CHO cells describing a direct comparison of temperature shifted versus non-temperature shifted cultures using 2D-DIGE. This investigation has enabled the identification of temperature-dependent as well as temperature-independent proteomic changes. 201 proteins were observed as differentially expressed following temperature shift, of which 118 were up regulated. Of the 53 proteins identified by MALDI-ToF MS, 23 were specifically differentially expressed upon reduction of the culture temperature and were found related to a variety of cellular functions such as regulation of growth (HNRPC), cap-independent translation (EIF4A), apoptosis (importin-alpha), the cytoskeleton (vimentin) and glycoprotein quality control (alpha glucosidase 2). CONCLUSION: These results indicate the extent of the temperature response in CHO-K1 cells and suggest a number of key regulatory proteins and pathways that are involved in modulating the response of cells to mild hypothermia. Regulation of these identified proteins and pathways could be useful for future approaches to engineer CHO cells for improved recombinant protein production.

Dataset Information

Ligand-dependent localization and intracellular stability of sigma-1 receptors in CHO-K1 cells.

Publications

Ligand-dependent localization and intracellular stability of sigma-1 receptors in CHO-K1 cells.

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