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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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Related in: MedlinePlus

Osteosarcoma cells overproduce OTR. Western-blot of OTR present in whole cell protein extracts obtained from HFF, MCF7, SaOS2 and U2OS cells, respectively. The total amount of protein loaded onto gels was adjusted to 93 g per each well. The blots were developed according described methods and subjected to quantitative analysis. The densitometric data indicate (Relative Densitometric Units, U) that both osteosarcoma cell lines tested produce as much as 3.6× more OTR as HFF cells and 20 more OTR in comparison to MCF-7 cells.
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fig13: Osteosarcoma cells overproduce OTR. Western-blot of OTR present in whole cell protein extracts obtained from HFF, MCF7, SaOS2 and U2OS cells, respectively. The total amount of protein loaded onto gels was adjusted to 93 g per each well. The blots were developed according described methods and subjected to quantitative analysis. The densitometric data indicate (Relative Densitometric Units, U) that both osteosarcoma cell lines tested produce as much as 3.6× more OTR as HFF cells and 20 more OTR in comparison to MCF-7 cells.

Mentions: The immunofluorescence observations of a variety of cells indicated quantitative differences in their content of intracellular OTR. The level of OTR antibody-mediated staining was consistently higher in osteosarcoma cells than in any of the other cells studied. We employed Western blotting to estimate the relative amount of OTR produced by various cell lines. When equal amounts of total protein were loaded onto SDS-PAGE gels, subsequent densitometric analysis of Western blots showed that osteosarcoma cells produce 3.6× as much OTR as HFF cells and 20× the amount produced by MCF7 breast cancer cells (Fig. 13). It appears, therefore, that the osteosarcoma cells not only exhibit a ligand-independent nuclear translocation mechanism, but also produce increased levels of the OTR in comparison to the non-bone tumours or normal cells examined.


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)

Osteosarcoma cells overproduce OTR. Western-blot of OTR present in whole cell protein extracts obtained from HFF, MCF7, SaOS2 and U2OS cells, respectively. The total amount of protein loaded onto gels was adjusted to 93 g per each well. The blots were developed according described methods and subjected to quantitative analysis. The densitometric data indicate (Relative Densitometric Units, U) that both osteosarcoma cell lines tested produce as much as 3.6× more OTR as HFF cells and 20 more OTR in comparison to MCF-7 cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig13: Osteosarcoma cells overproduce OTR. Western-blot of OTR present in whole cell protein extracts obtained from HFF, MCF7, SaOS2 and U2OS cells, respectively. The total amount of protein loaded onto gels was adjusted to 93 g per each well. The blots were developed according described methods and subjected to quantitative analysis. The densitometric data indicate (Relative Densitometric Units, U) that both osteosarcoma cell lines tested produce as much as 3.6× more OTR as HFF cells and 20 more OTR in comparison to MCF-7 cells.
Mentions: The immunofluorescence observations of a variety of cells indicated quantitative differences in their content of intracellular OTR. The level of OTR antibody-mediated staining was consistently higher in osteosarcoma cells than in any of the other cells studied. We employed Western blotting to estimate the relative amount of OTR produced by various cell lines. When equal amounts of total protein were loaded onto SDS-PAGE gels, subsequent densitometric analysis of Western blots showed that osteosarcoma cells produce 3.6× as much OTR as HFF cells and 20× the amount produced by MCF7 breast cancer cells (Fig. 13). It appears, therefore, that the osteosarcoma cells not only exhibit a ligand-independent nuclear translocation mechanism, but also produce increased levels of the OTR in comparison to the non-bone tumours or normal cells examined.

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