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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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Nucleolar localization of OTR in MCF-7 and MG-63 cells. Standard epifluorescent immunocytochemistry (A) and DIC phase contrast microscopy (B) images of the same group of MCF-7 cells show distinct nucleolar localization of OTR. MCF-7 cells were stained with anti-OTR MAb and a FITC-conjugated rabbit anti-mouse IgG (A). Images C and D represent midsection confocal microscopy of MG63 osteosarcoma cells stained with the same antibodies as above. Control cells (C) were not treated with OT, while the cells in image D were treated with 10−7 M OT for 15 min.
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fig12: Nucleolar localization of OTR in MCF-7 and MG-63 cells. Standard epifluorescent immunocytochemistry (A) and DIC phase contrast microscopy (B) images of the same group of MCF-7 cells show distinct nucleolar localization of OTR. MCF-7 cells were stained with anti-OTR MAb and a FITC-conjugated rabbit anti-mouse IgG (A). Images C and D represent midsection confocal microscopy of MG63 osteosarcoma cells stained with the same antibodies as above. Control cells (C) were not treated with OT, while the cells in image D were treated with 10−7 M OT for 15 min.

Mentions: The OTR localization appears to be in several regions of the nucleoplasm of HFF cells. The retention of both OTR-GFP and native OTR within the nucleus last hours to days (Fig. 3). In nearly all images examined, the OTR (or OTR-GFP) could be seen in the nuclei of cells as specks (4–20 in number), with ill-defined margins. The large specks are surrounded by smaller antibody-positive dots too numerous to be enumerated (Fig. 10A) and are near the geometrical midline of the nucleus (Fig. 10B); in no instance is the OTR associated with the internal nuclear envelope.The latter is further documented by double-staining the cells for lamin B and OTR (Fig. 11). In some instances, OTR was distinctly localized into at least one of the nucleoli (Fig. 12A–B) as demonstrated by superimposing the DIC and fluorescence images of the same field. It appeared that the nucleoli staining occurred prior to treatment with a ligand (Fig. 12C—D). The reason for localization of OTR to some of the nucleoli in some cells, and in certain cases, to all nucleoli of the same cell, is not known.


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)

Nucleolar localization of OTR in MCF-7 and MG-63 cells. Standard epifluorescent immunocytochemistry (A) and DIC phase contrast microscopy (B) images of the same group of MCF-7 cells show distinct nucleolar localization of OTR. MCF-7 cells were stained with anti-OTR MAb and a FITC-conjugated rabbit anti-mouse IgG (A). Images C and D represent midsection confocal microscopy of MG63 osteosarcoma cells stained with the same antibodies as above. Control cells (C) were not treated with OT, while the cells in image D were treated with 10−7 M OT for 15 min.
© Copyright Policy
Related In: Results  -  Collection

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

fig12: Nucleolar localization of OTR in MCF-7 and MG-63 cells. Standard epifluorescent immunocytochemistry (A) and DIC phase contrast microscopy (B) images of the same group of MCF-7 cells show distinct nucleolar localization of OTR. MCF-7 cells were stained with anti-OTR MAb and a FITC-conjugated rabbit anti-mouse IgG (A). Images C and D represent midsection confocal microscopy of MG63 osteosarcoma cells stained with the same antibodies as above. Control cells (C) were not treated with OT, while the cells in image D were treated with 10−7 M OT for 15 min.
Mentions: The OTR localization appears to be in several regions of the nucleoplasm of HFF cells. The retention of both OTR-GFP and native OTR within the nucleus last hours to days (Fig. 3). In nearly all images examined, the OTR (or OTR-GFP) could be seen in the nuclei of cells as specks (4–20 in number), with ill-defined margins. The large specks are surrounded by smaller antibody-positive dots too numerous to be enumerated (Fig. 10A) and are near the geometrical midline of the nucleus (Fig. 10B); in no instance is the OTR associated with the internal nuclear envelope.The latter is further documented by double-staining the cells for lamin B and OTR (Fig. 11). In some instances, OTR was distinctly localized into at least one of the nucleoli (Fig. 12A–B) as demonstrated by superimposing the DIC and fluorescence images of the same field. It appeared that the nucleoli staining occurred prior to treatment with a ligand (Fig. 12C—D). The reason for localization of OTR to some of the nucleoli in some cells, and in certain cases, to all nucleoli of the same cell, is not known.

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