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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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Midsection confocal microscopyimages of primary human fibroblast(HFF) cells. Images A through C wereobtained from control (OT-untreatedcultures) while images D through F were from cultures treated with OT at10−7 M for 15 min. Images A and Drepresent staining with propidiumiodide, images B and E are immuno-histochemistry using monoclonal OTR-antibody and FITC conjugated rabbitanti-mouse IgG, and images C and Fare overlays of images A and B, and Dand E, respectively.
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fig06: Midsection confocal microscopyimages of primary human fibroblast(HFF) cells. Images A through C wereobtained from control (OT-untreatedcultures) while images D through F were from cultures treated with OT at10−7 M for 15 min. Images A and Drepresent staining with propidiumiodide, images B and E are immuno-histochemistry using monoclonal OTR-antibody and FITC conjugated rabbitanti-mouse IgG, and images C and Fare overlays of images A and B, and Dand E, respectively.

Mentions: Immunofluorescence avoids problems associated with expression of the OTR-GFP gene, particularly the presence of the overexpressed protein in the cell, time required for expression of the fusion protein, and possible interference of GFP with either the translocation of OTR across the nuclear pore or the interaction of OTR with karyophylic proteins. Similarly to OTR-GFP experiments, immunofluorescence studies of endogenous OTR in MCF7 cells show that both internalization and nuclear localization of the OTR is strictly dependent on treatment of cells with ligand (Fig. 4). In MCF7 cells, the OT-dependent nucleoplasmic transport of OTR can be observed within minutes following the addition of ligand. However, only a minor portion of total cytoplasmic OTR enters the nucleus, while most of the label accumulates in the perinuclear region. Once transported into the nucleus, the OTR remained within the nucleus regardless of the extracellular presence of OT for at least several hours (results not shown). Conversely, the endogenous OTR localize into the nuclei of bone cancer derived U2OS cells (Fig. 5A–C) and SaOS2 cells (Fig. 5D–E) regardless of OT treatment. Nuclear transport of OTR in normal human fibroblasts (HFF) cells is only partially dependent on exogenous OT; however, its nuclear localization is greatly enhanced by OT treatment (Fig. 6).


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)

Midsection confocal microscopyimages of primary human fibroblast(HFF) cells. Images A through C wereobtained from control (OT-untreatedcultures) while images D through F were from cultures treated with OT at10−7 M for 15 min. Images A and Drepresent staining with propidiumiodide, images B and E are immuno-histochemistry using monoclonal OTR-antibody and FITC conjugated rabbitanti-mouse IgG, and images C and Fare overlays of images A and B, and Dand E, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig06: Midsection confocal microscopyimages of primary human fibroblast(HFF) cells. Images A through C wereobtained from control (OT-untreatedcultures) while images D through F were from cultures treated with OT at10−7 M for 15 min. Images A and Drepresent staining with propidiumiodide, images B and E are immuno-histochemistry using monoclonal OTR-antibody and FITC conjugated rabbitanti-mouse IgG, and images C and Fare overlays of images A and B, and Dand E, respectively.
Mentions: Immunofluorescence avoids problems associated with expression of the OTR-GFP gene, particularly the presence of the overexpressed protein in the cell, time required for expression of the fusion protein, and possible interference of GFP with either the translocation of OTR across the nuclear pore or the interaction of OTR with karyophylic proteins. Similarly to OTR-GFP experiments, immunofluorescence studies of endogenous OTR in MCF7 cells show that both internalization and nuclear localization of the OTR is strictly dependent on treatment of cells with ligand (Fig. 4). In MCF7 cells, the OT-dependent nucleoplasmic transport of OTR can be observed within minutes following the addition of ligand. However, only a minor portion of total cytoplasmic OTR enters the nucleus, while most of the label accumulates in the perinuclear region. Once transported into the nucleus, the OTR remained within the nucleus regardless of the extracellular presence of OT for at least several hours (results not shown). Conversely, the endogenous OTR localize into the nuclei of bone cancer derived U2OS cells (Fig. 5A–C) and SaOS2 cells (Fig. 5D–E) regardless of OT treatment. Nuclear transport of OTR in normal human fibroblasts (HFF) cells is only partially dependent on exogenous OT; however, its nuclear localization is greatly enhanced by OT treatment (Fig. 6).

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