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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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Association of OTR with the perinuclear and nuclear fractions of MCF-7 cells. A–C depict control cells and D–F represent OT-treated (10−7 M for 15 min) cells. A and D are phase contrast images, B through F are confocal images of immunofluorescence with OTR-Ab, FITC-labeled conjugate and propidium iodide. B and E are stacked confocal images while C and F are a single midsection images.
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fig09: Association of OTR with the perinuclear and nuclear fractions of MCF-7 cells. A–C depict control cells and D–F represent OT-treated (10−7 M for 15 min) cells. A and D are phase contrast images, B through F are confocal images of immunofluorescence with OTR-Ab, FITC-labeled conjugate and propidium iodide. B and E are stacked confocal images while C and F are a single midsection images.

Mentions: We set out to directly determine to what extent the material reacting with the OTR-antibody remains associated with isolated nuclei. In order to highlight this perinuclear component, we used an abbreviated nuclear isolation procedure designed to obtain nuclei with an excess of the nuclear-associated cytoplasmic fragments (crude nuclei; procedure 2 in ‘Methods’). Nuclear preparations from OT-treated and -untreated MCF7 cells were fixed and subjected to immunofluorescence confocal microscopy. The results showed that nuclei of MCF7 cells lack OTR in the absence of OT treatment (Fig. 9). There was, however, a significant increase of OTR within nuclei as well as the perinuclear cytoplasm following OT treatment.


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)

Association of OTR with the perinuclear and nuclear fractions of MCF-7 cells. A–C depict control cells and D–F represent OT-treated (10−7 M for 15 min) cells. A and D are phase contrast images, B through F are confocal images of immunofluorescence with OTR-Ab, FITC-labeled conjugate and propidium iodide. B and E are stacked confocal images while C and F are a single midsection images.
© Copyright Policy
Related In: Results  -  Collection

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

fig09: Association of OTR with the perinuclear and nuclear fractions of MCF-7 cells. A–C depict control cells and D–F represent OT-treated (10−7 M for 15 min) cells. A and D are phase contrast images, B through F are confocal images of immunofluorescence with OTR-Ab, FITC-labeled conjugate and propidium iodide. B and E are stacked confocal images while C and F are a single midsection images.
Mentions: We set out to directly determine to what extent the material reacting with the OTR-antibody remains associated with isolated nuclei. In order to highlight this perinuclear component, we used an abbreviated nuclear isolation procedure designed to obtain nuclei with an excess of the nuclear-associated cytoplasmic fragments (crude nuclei; procedure 2 in ‘Methods’). Nuclear preparations from OT-treated and -untreated MCF7 cells were fixed and subjected to immunofluorescence confocal microscopy. The results showed that nuclei of MCF7 cells lack OTR in the absence of OT treatment (Fig. 9). There was, however, a significant increase of OTR within nuclei as well as the perinuclear cytoplasm following OT treatment.

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