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Constitutive and ligand-induced nuclear localization of oxytocin receptor.

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.

SUBMITTER: Kinsey CG

PROVIDER: S-EPMC4401223 | biostudies-literature | 2007 Jan-Feb

REPOSITORIES: biostudies-literature

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Constitutive and ligand-induced nuclear localization of oxytocin receptor.

Kinsey Conan G CG Bussolati Gianni G Bosco Martino M Kimura Tadashi T Pizzorno Marie C MC Chernin Mitchell I MI Cassoni Paola P Novak Josef F JF

Journal of cellular and molecular medicine 20070101 1

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 con ...[more]

PMID: 17367504

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Project description:The cannabinoid CB(1) (CB(1)) and dopamine D(2) (D(2)) receptors are coexpressed in the basal ganglia, an area of the brain involved in such processes as cognition, motor function, and emotional control. Several lines of evidence suggest that CB(1) and D(2) receptors may oligomerize, providing a unique pharmacology in vitro and in vivo. However, limited information exists on the regulation of CB(1) and D(2) receptor dimers. We used a novel technique, multicolor bimolecular fluorescence complementation (MBiFC) to examine the subcellular localization of CB(1)-D(2L) heterodimers as well as D(2L)-D(2L) homodimers in a neuronal cell model, Cath. a differentiated cells. MBiFC was then used to explore the effects of persistent ligand treatment on receptor dimerization at the plasma membrane and intracellularly. Persistent (20-h) agonist treatment resulted in increased formation of CB(1)-D(2L) heterodimers relative to the D(2L)-D(2L) homodimers. The effects of the D(2) agonist quinpirole were restricted to the intracellular compartment and may reflect increased D(2L) receptor expression. In contrast, treatment with the CB(1) receptor agonist (2)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP55, 940) produced increases in both membrane and intracellular CB(1)-D(2L) heterodimers independently of alterations in CB(1) receptor expression. The effects of CB(1) receptor activation were attenuated by the CB(1) antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) and were both time- and dose-dependent. The effects of CB(1) activation were examined further by combining MBiFC with a constitutively active CB(1) receptor mutant, CB(1)T210I. These studies demonstrated that the expression of CB(1)T210I increased intracellular CB(1)-D(2L) heterodimer formation. In summary, agonist-induced modulation of CB(1)-D(2L) oligomerization may have physiological implications in diseases such as Parkinson's disease and drug abuse.

Dataset Information

Constitutive and ligand-induced nuclear localization of oxytocin receptor.

Publications

Constitutive and ligand-induced nuclear localization of oxytocin receptor.

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