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Constitutive and ligand-induced nuclear localization of oxytocin receptor.

Kinsey CG, Bussolati G, Bosco M, Kimura T, Pizzorno MC, Chernin MI, Cassoni P, Novak JF - J. Cell. Mol. Med. (2007 Jan-Feb)

Bottom Line: Oxytocin receptor (OTR) is a membrane protein known to mediate oxytocin (OT) effects, in both normal and neoplastic cells.Treatment with oxytocin causes internalization of OTR and the resulting vesicles accumulate in the vicinity of the nucleus and some of the perinuclear OTR enters the nucleus.The evidence of OTR compartmentalization at the cell nucleus (either ligand-dependent or constitutive) in different cell types suggests still unknown biological functions of this protein or its ligand and adds this G-protein-coupled receptor to other heptahelical receptors displaying this atypical and unexpected nuclear localization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Bucknell University, Lewisburg, PA, USA.

ABSTRACT
Oxytocin receptor (OTR) is a membrane protein known to mediate oxytocin (OT) effects, in both normal and neoplastic cells. We report here that human osteosarcoma (U2OS, MG63, OS15 and SaOS2), breast cancer (MCF7), and primary human fibroblastic cells (HFF) all exhibit OTR not only on the cell membrane, but also in the various nuclear compartments including the nucleolus. Both an OTR-GFP fusion protein and the native OTR appear to be localized to the nucleus as detected by transfection and/or confocal immunofluorescence, respectively. Treatment with oxytocin causes internalization of OTR and the resulting vesicles accumulate in the vicinity of the nucleus and some of the perinuclear OTR enters the nucleus. Western blots indicate that OTR in the nucleus and on the plasma membrane are likely to be the same biochemical and immunological entities. It appears that OTR is first visible in the nucleoli and subsequently disperses within the nucleus into 4-20 spots while some of the OTR diffuses throughout the nucleoplasm. The behaviour and kinetics of OTR-GFP and OTR are different, indicating interference by GFP in both OTR entrance into the nucleus and subsequent relocalization of OTR within the nucleus. There are important differences among the tested cells, such as the requirement of a ligand for transfer of OTR in nuclei. A constitutive internalization of OTR was found only in osteosarcoma cells, while the nuclear localization in all other tested cells was dependent on ligand binding. The amount of OTR-positive material within and in the vicinity of the nucleus increased following a treatment with oxytocin in both constitutive and ligand-dependent type of cells. The evidence of OTR compartmentalization at the cell nucleus (either ligand-dependent or constitutive) in different cell types suggests still unknown biological functions of this protein or its ligand and adds this G-protein-coupled receptor to other heptahelical receptors displaying this atypical and unexpected nuclear localization.

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Constitutive and time-dependent translocation of OTR-GFP into the nuclei of U2OS cells. Image A represents midsection confocal microscopy of the untreated cells, 2 days post-transfection with pEGFP-N3-OTR (time 0). Cells in images B through F were all treated with OT (10−7 M) for 6, 24, 48, 72 and 96 hrs, respectively.
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fig03: Constitutive and time-dependent translocation of OTR-GFP into the nuclei of U2OS cells. Image A represents midsection confocal microscopy of the untreated cells, 2 days post-transfection with pEGFP-N3-OTR (time 0). Cells in images B through F were all treated with OT (10−7 M) for 6, 24, 48, 72 and 96 hrs, respectively.

Mentions: In contrast to MCF-7 cells, the OTR-GFP protein in U2OS osteosarcoma cells exhibited strong staining within the nucleus even in the absence of OT treatment (Fig. 3A). OT treatment of U2OS cells resulted in increased vesiculation of OTR-GFP protein. The vesicles surrounded the nucleus followed by a time-dependent increase of the amount in fluorescence within the nucleus (Fig. 3B–E). In other experiments we found a number of other osteosarcoma cell lines (MG-63, SaOS2 and OS-15) localize OTR-GFP into the nucleus with or without OT treatment (see below). The time-point of initial localization of OTR-GFP into the nucleus in osteosarcoma cells is difficult to assess as the expression and translocation of the fusion protein overlaps with the transfection procedure itself. Regardless, transfection experiments revealed a difference between MCF7 breast carcinoma and a number of osteosarcoma cell lines in terms of OTR-GFP sub-cellular localization and responses to the treatment with a ligand. It is also possible that the slow kinetics of GFP-OTR nuclear localization in MCF-7 cells was due to a cell-specific interference by GFP in the translocation of OTR-GFP. In order to resolve this possibility, the localization of endogenous OTR within cells was examined using a monoclonal antibody specific for the OTR.


Constitutive and ligand-induced nuclear localization of oxytocin receptor.

Kinsey CG, Bussolati G, Bosco M, Kimura T, Pizzorno MC, Chernin MI, Cassoni P, Novak JF - J. Cell. Mol. Med. (2007 Jan-Feb)

Constitutive and time-dependent translocation of OTR-GFP into the nuclei of U2OS cells. Image A represents midsection confocal microscopy of the untreated cells, 2 days post-transfection with pEGFP-N3-OTR (time 0). Cells in images B through F were all treated with OT (10−7 M) for 6, 24, 48, 72 and 96 hrs, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401223&req=5

fig03: Constitutive and time-dependent translocation of OTR-GFP into the nuclei of U2OS cells. Image A represents midsection confocal microscopy of the untreated cells, 2 days post-transfection with pEGFP-N3-OTR (time 0). Cells in images B through F were all treated with OT (10−7 M) for 6, 24, 48, 72 and 96 hrs, respectively.
Mentions: In contrast to MCF-7 cells, the OTR-GFP protein in U2OS osteosarcoma cells exhibited strong staining within the nucleus even in the absence of OT treatment (Fig. 3A). OT treatment of U2OS cells resulted in increased vesiculation of OTR-GFP protein. The vesicles surrounded the nucleus followed by a time-dependent increase of the amount in fluorescence within the nucleus (Fig. 3B–E). In other experiments we found a number of other osteosarcoma cell lines (MG-63, SaOS2 and OS-15) localize OTR-GFP into the nucleus with or without OT treatment (see below). The time-point of initial localization of OTR-GFP into the nucleus in osteosarcoma cells is difficult to assess as the expression and translocation of the fusion protein overlaps with the transfection procedure itself. Regardless, transfection experiments revealed a difference between MCF7 breast carcinoma and a number of osteosarcoma cell lines in terms of OTR-GFP sub-cellular localization and responses to the treatment with a ligand. It is also possible that the slow kinetics of GFP-OTR nuclear localization in MCF-7 cells was due to a cell-specific interference by GFP in the translocation of OTR-GFP. In order to resolve this possibility, the localization of endogenous OTR within cells was examined using a monoclonal antibody specific for the OTR.

Bottom Line: Oxytocin receptor (OTR) is a membrane protein known to mediate oxytocin (OT) effects, in both normal and neoplastic cells.Treatment with oxytocin causes internalization of OTR and the resulting vesicles accumulate in the vicinity of the nucleus and some of the perinuclear OTR enters the nucleus.The evidence of OTR compartmentalization at the cell nucleus (either ligand-dependent or constitutive) in different cell types suggests still unknown biological functions of this protein or its ligand and adds this G-protein-coupled receptor to other heptahelical receptors displaying this atypical and unexpected nuclear localization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Bucknell University, Lewisburg, PA, USA.

ABSTRACT
Oxytocin receptor (OTR) is a membrane protein known to mediate oxytocin (OT) effects, in both normal and neoplastic cells. We report here that human osteosarcoma (U2OS, MG63, OS15 and SaOS2), breast cancer (MCF7), and primary human fibroblastic cells (HFF) all exhibit OTR not only on the cell membrane, but also in the various nuclear compartments including the nucleolus. Both an OTR-GFP fusion protein and the native OTR appear to be localized to the nucleus as detected by transfection and/or confocal immunofluorescence, respectively. Treatment with oxytocin causes internalization of OTR and the resulting vesicles accumulate in the vicinity of the nucleus and some of the perinuclear OTR enters the nucleus. Western blots indicate that OTR in the nucleus and on the plasma membrane are likely to be the same biochemical and immunological entities. It appears that OTR is first visible in the nucleoli and subsequently disperses within the nucleus into 4-20 spots while some of the OTR diffuses throughout the nucleoplasm. The behaviour and kinetics of OTR-GFP and OTR are different, indicating interference by GFP in both OTR entrance into the nucleus and subsequent relocalization of OTR within the nucleus. There are important differences among the tested cells, such as the requirement of a ligand for transfer of OTR in nuclei. A constitutive internalization of OTR was found only in osteosarcoma cells, while the nuclear localization in all other tested cells was dependent on ligand binding. The amount of OTR-positive material within and in the vicinity of the nucleus increased following a treatment with oxytocin in both constitutive and ligand-dependent type of cells. The evidence of OTR compartmentalization at the cell nucleus (either ligand-dependent or constitutive) in different cell types suggests still unknown biological functions of this protein or its ligand and adds this G-protein-coupled receptor to other heptahelical receptors displaying this atypical and unexpected nuclear localization.

Show MeSH
Related in: MedlinePlus