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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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Cell-surface ligand is internalized by OT (oxytocin)-treated cells. (A) Acid-wash method using 125I-oxytocin. For OT internalization by the acid-wash technique, confluent cultures of U2OS cells were washed and cooled to 4°C before adding 125I-OT and cold OT to a final concentration of 10−9 and 10−5 M, respectively. The binding was equilibrated at 4°C for 4 hrs. At the end of the binding period, the cells were transferred to 37°C for the indicated time periods. The cells were washed, treated with acid wash and finally dissolved (see ‘Methods’). Both acid wash (representing the 125I-oxytocin fraction bound at the surface) and the cell digest (representing the 125I-oxytocin fraction internalized) were collected and their radioactivity determined. Lines are the mean of two different experiments. Standard deviation were less than 10%. (B) Flow cytometry method using a specific antibody to human OTR. Flow cytometry of OT internalization was performed using cells equilibrated with 10−9 M OT at 4°C. The hormone-equilibrated cells were then incubated for additional 30 min either at 4 or 37°C. The cell surface OTR was labeled sequentially with 1F3 MAb and FITC-secondary antibody. The cells were scrapped and analysed by means of flow cytometry.
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fig01: Cell-surface ligand is internalized by OT (oxytocin)-treated cells. (A) Acid-wash method using 125I-oxytocin. For OT internalization by the acid-wash technique, confluent cultures of U2OS cells were washed and cooled to 4°C before adding 125I-OT and cold OT to a final concentration of 10−9 and 10−5 M, respectively. The binding was equilibrated at 4°C for 4 hrs. At the end of the binding period, the cells were transferred to 37°C for the indicated time periods. The cells were washed, treated with acid wash and finally dissolved (see ‘Methods’). Both acid wash (representing the 125I-oxytocin fraction bound at the surface) and the cell digest (representing the 125I-oxytocin fraction internalized) were collected and their radioactivity determined. Lines are the mean of two different experiments. Standard deviation were less than 10%. (B) Flow cytometry method using a specific antibody to human OTR. Flow cytometry of OT internalization was performed using cells equilibrated with 10−9 M OT at 4°C. The hormone-equilibrated cells were then incubated for additional 30 min either at 4 or 37°C. The cell surface OTR was labeled sequentially with 1F3 MAb and FITC-secondary antibody. The cells were scrapped and analysed by means of flow cytometry.

Mentions: U2OS osteosarcoma cells were equilibrated with 10−9 M extracellular 125I-OT at 4°C and the loss of cell surface label was followed in a time-dependent manner in the presence or absence of excess OT. By the acid wash technique, it was established that approximately 85% of total cell surface-localized 125I-OT is internalized within the first 20 min after ligand addition (Fig. 1A). These data suggest that the internalization of cell-surface bound radioactive OT begins immediately following the receptor ligand binding.


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)

Cell-surface ligand is internalized by OT (oxytocin)-treated cells. (A) Acid-wash method using 125I-oxytocin. For OT internalization by the acid-wash technique, confluent cultures of U2OS cells were washed and cooled to 4°C before adding 125I-OT and cold OT to a final concentration of 10−9 and 10−5 M, respectively. The binding was equilibrated at 4°C for 4 hrs. At the end of the binding period, the cells were transferred to 37°C for the indicated time periods. The cells were washed, treated with acid wash and finally dissolved (see ‘Methods’). Both acid wash (representing the 125I-oxytocin fraction bound at the surface) and the cell digest (representing the 125I-oxytocin fraction internalized) were collected and their radioactivity determined. Lines are the mean of two different experiments. Standard deviation were less than 10%. (B) Flow cytometry method using a specific antibody to human OTR. Flow cytometry of OT internalization was performed using cells equilibrated with 10−9 M OT at 4°C. The hormone-equilibrated cells were then incubated for additional 30 min either at 4 or 37°C. The cell surface OTR was labeled sequentially with 1F3 MAb and FITC-secondary antibody. The cells were scrapped and analysed by means of flow cytometry.
© Copyright Policy
Related In: Results  -  Collection

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

fig01: Cell-surface ligand is internalized by OT (oxytocin)-treated cells. (A) Acid-wash method using 125I-oxytocin. For OT internalization by the acid-wash technique, confluent cultures of U2OS cells were washed and cooled to 4°C before adding 125I-OT and cold OT to a final concentration of 10−9 and 10−5 M, respectively. The binding was equilibrated at 4°C for 4 hrs. At the end of the binding period, the cells were transferred to 37°C for the indicated time periods. The cells were washed, treated with acid wash and finally dissolved (see ‘Methods’). Both acid wash (representing the 125I-oxytocin fraction bound at the surface) and the cell digest (representing the 125I-oxytocin fraction internalized) were collected and their radioactivity determined. Lines are the mean of two different experiments. Standard deviation were less than 10%. (B) Flow cytometry method using a specific antibody to human OTR. Flow cytometry of OT internalization was performed using cells equilibrated with 10−9 M OT at 4°C. The hormone-equilibrated cells were then incubated for additional 30 min either at 4 or 37°C. The cell surface OTR was labeled sequentially with 1F3 MAb and FITC-secondary antibody. The cells were scrapped and analysed by means of flow cytometry.
Mentions: U2OS osteosarcoma cells were equilibrated with 10−9 M extracellular 125I-OT at 4°C and the loss of cell surface label was followed in a time-dependent manner in the presence or absence of excess OT. By the acid wash technique, it was established that approximately 85% of total cell surface-localized 125I-OT is internalized within the first 20 min after ligand addition (Fig. 1A). These data suggest that the internalization of cell-surface bound radioactive OT begins immediately following the receptor ligand binding.

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