Constitutive and ligand-induced nuclear localization of oxytocin receptor.
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.
Kinsey
Conan G
CG
Department of Biology, Bucknell University, Lewisburg, PA, USA.
Bussolati
Gianni
G
Bosco
Martino
M
Kimura
Tadashi
T
Pizzorno
Marie C
MC
Chernin
Mitchell I
MI
Cassoni
Paola
P
Novak
Josef F
JF
D001943:Breast Neoplasms / Q000473:pathology; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008024:Ligands; D018613:Microscopy, Confocal; D012516:Osteosarcoma / Q000473:pathology; D010121:Oxytocin / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D018045:Receptors, Oxytocin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D005456:Fluorescent Dyes; D008024:Ligands; D018045:Receptors, Oxytocin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010121:Oxytocin; D016650:Fluorescein-5-isothiocyanate