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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

Treatment with OT increases translocation of OTR into nuclei of HFF cells. Western-blot indicating the presence of the OTR in nuclear protein fractions (procedure 1, ‘Methods’) of both control and OT-treated (10−7 M, 15 min) HFF cells. The relative densitometric units (U) per microgram of protein indicate approximately 10 times higher level of OTR in OT-treated cells. The total amount of protein loaded onto the wells is as follows: whole cell extract (60.75 g), nuclear extract (54 g) and nuclear extract from cells treated with OT (24 g).
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fig08: Treatment with OT increases translocation of OTR into nuclei of HFF cells. Western-blot indicating the presence of the OTR in nuclear protein fractions (procedure 1, ‘Methods’) of both control and OT-treated (10−7 M, 15 min) HFF cells. The relative densitometric units (U) per microgram of protein indicate approximately 10 times higher level of OTR in OT-treated cells. The total amount of protein loaded onto the wells is as follows: whole cell extract (60.75 g), nuclear extract (54 g) and nuclear extract from cells treated with OT (24 g).

Mentions: Whole cell protein preparations and nuclear protein extracts of U2OS osteosarcoma cells were prepared and separated on SDS-polyacrylamide gels. Western blots revealed the expected ∼59 kD OTR in whole cell extracts as well as nuclear fractions (Fig. 7). Only about 6% of total OTR present in the cell membrane could be seen in the nuclear fraction of HFF cells; however, this amount increased fourfold following treatment with OT (Fig. 8). These results are in keeping with constitutive and ligand-induced accumulation of OTR in the proximity of the nucleus as indicated by fluorescence microscopy. Thus, the ‘nuclear fractions’ analysed by Western blot could include OTR isolated from the vicinity of the nucleus as well as that localized exclusively within the nucleoplasm.


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)

Treatment with OT increases translocation of OTR into nuclei of HFF cells. Western-blot indicating the presence of the OTR in nuclear protein fractions (procedure 1, ‘Methods’) of both control and OT-treated (10−7 M, 15 min) HFF cells. The relative densitometric units (U) per microgram of protein indicate approximately 10 times higher level of OTR in OT-treated cells. The total amount of protein loaded onto the wells is as follows: whole cell extract (60.75 g), nuclear extract (54 g) and nuclear extract from cells treated with OT (24 g).
© Copyright Policy
Related In: Results  -  Collection

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

fig08: Treatment with OT increases translocation of OTR into nuclei of HFF cells. Western-blot indicating the presence of the OTR in nuclear protein fractions (procedure 1, ‘Methods’) of both control and OT-treated (10−7 M, 15 min) HFF cells. The relative densitometric units (U) per microgram of protein indicate approximately 10 times higher level of OTR in OT-treated cells. The total amount of protein loaded onto the wells is as follows: whole cell extract (60.75 g), nuclear extract (54 g) and nuclear extract from cells treated with OT (24 g).
Mentions: Whole cell protein preparations and nuclear protein extracts of U2OS osteosarcoma cells were prepared and separated on SDS-polyacrylamide gels. Western blots revealed the expected ∼59 kD OTR in whole cell extracts as well as nuclear fractions (Fig. 7). Only about 6% of total OTR present in the cell membrane could be seen in the nuclear fraction of HFF cells; however, this amount increased fourfold following treatment with OT (Fig. 8). These results are in keeping with constitutive and ligand-induced accumulation of OTR in the proximity of the nucleus as indicated by fluorescence microscopy. Thus, the ‘nuclear fractions’ analysed by Western blot could include OTR isolated from the vicinity of the nucleus as well as that localized exclusively within the nucleoplasm.

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