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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 microscopy of OTR-GFP in pEGFP-N3-OTR-transfected MCF7 cells before (A) and after (B–D) OT treatment. Cells in images (B–D) were treated with OT (10−7 M) for 15 min, 24 and 48 hrs, respectively. While the control nuclei () are essentially free of the GFP-mediated fluorescence, their intensity increased with length of OT treatment.
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fig02: Midsection confocal microscopy of OTR-GFP in pEGFP-N3-OTR-transfected MCF7 cells before (A) and after (B–D) OT treatment. Cells in images (B–D) were treated with OT (10−7 M) for 15 min, 24 and 48 hrs, respectively. While the control nuclei () are essentially free of the GFP-mediated fluorescence, their intensity increased with length of OT treatment.

Mentions: The functionality of the OTR-GFP fusion protein was ascertained by cytological relocalization of the expressed fusion protein following treatment of cells with OT. Midsection confocal microscopy images of transfected MCF-7 cells are shown in Fig. 2. Prior to the treatment with OT, the OTR-GFP fusion protein appears equally distributed between cell surface and cytoplasm with essentially no fluorescence in the nucleus (Fig. 2A). For translocation of OTR-GFP from cell membrane/cytoplasm to nuclei, the MCF-7 cells required treatment with exogenous OT (10−7M).Fifteen minutes after treatment with OT (Fig. 2B) nearly all of the OTR-GFP was sequestered into cytoplasmic vesicles, which appeared equally distributed throughout the cytoplasm. Continuous 24-hrs treatment with OT (Fig. 2C) resulted in concentration of the granular fluorescence around the nucleus with some accumulation of GFP-generated fluorescence within the nucleus itself. After 48 hrs of continuous OT treatment, the MCF7 cells exhibited large OTR-containing vesicles within the cytoplasm (Fig. 2D), some accumulation around the nucleus and a distinct localization of fluorescence within the nuclei of some but not of all the cells. In control experiments transfected with GFP plasmid alone, the fluorescence became evenly distributed within the cytoplasm and nucleus soon after the expression of the GFP. The process of GFP distribution was not affected by a treatment with OT (results not shown).


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 microscopy of OTR-GFP in pEGFP-N3-OTR-transfected MCF7 cells before (A) and after (B–D) OT treatment. Cells in images (B–D) were treated with OT (10−7 M) for 15 min, 24 and 48 hrs, respectively. While the control nuclei () are essentially free of the GFP-mediated fluorescence, their intensity increased with length of OT treatment.
© Copyright Policy
Related In: Results  -  Collection

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

fig02: Midsection confocal microscopy of OTR-GFP in pEGFP-N3-OTR-transfected MCF7 cells before (A) and after (B–D) OT treatment. Cells in images (B–D) were treated with OT (10−7 M) for 15 min, 24 and 48 hrs, respectively. While the control nuclei () are essentially free of the GFP-mediated fluorescence, their intensity increased with length of OT treatment.
Mentions: The functionality of the OTR-GFP fusion protein was ascertained by cytological relocalization of the expressed fusion protein following treatment of cells with OT. Midsection confocal microscopy images of transfected MCF-7 cells are shown in Fig. 2. Prior to the treatment with OT, the OTR-GFP fusion protein appears equally distributed between cell surface and cytoplasm with essentially no fluorescence in the nucleus (Fig. 2A). For translocation of OTR-GFP from cell membrane/cytoplasm to nuclei, the MCF-7 cells required treatment with exogenous OT (10−7M).Fifteen minutes after treatment with OT (Fig. 2B) nearly all of the OTR-GFP was sequestered into cytoplasmic vesicles, which appeared equally distributed throughout the cytoplasm. Continuous 24-hrs treatment with OT (Fig. 2C) resulted in concentration of the granular fluorescence around the nucleus with some accumulation of GFP-generated fluorescence within the nucleus itself. After 48 hrs of continuous OT treatment, the MCF7 cells exhibited large OTR-containing vesicles within the cytoplasm (Fig. 2D), some accumulation around the nucleus and a distinct localization of fluorescence within the nuclei of some but not of all the cells. In control experiments transfected with GFP plasmid alone, the fluorescence became evenly distributed within the cytoplasm and nucleus soon after the expression of the GFP. The process of GFP distribution was not affected by a treatment with OT (results not shown).

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