engleski

Localization of dopamine receptors in a neuroblastoma cell line

Human neuroblastoma cell line SH-SY5Y is a dopaminergic neuronal cell line expressing tyrosin-hydroxoylase, vesicular monoamine transporter VMAT2 and dopamine receptors (DR). It is a widely used model to study mechanisms of neurodegeneration, dopamine toxicity and DR ligands. However, there is no report about the expression and the subcellular localization of DR in these cells. MATERIALS AND METHODS: SH-SY5Y cell line was terminally differentiated into neuron-like cells after 5-day treatment with retinoic acid (10 microM), followed by 3-day treatment with brain-derived neurotropic factor (BDNF, 50ng/mL). Cells were fixed with 4% paraformaldehyde and stained using novel anti-DRD2, anti-DRD3 and anti-DRD4 monoclonal antibodies. The specificity of the antibodies used has been previously characterized by Wolstencroft et al. (2007). RESULTS: We found strong expression of DRD2 along the cell membrane, but also in cytoplasmatic vesicles. The expression of DRD3 was significantly weaker with almost no vesicle-like reactivity. We observed no DRD4 immunoreactivity in this cell line. CONCLUSIONS: We showed an expected membrane localization, but also a significant endosomal localization of DRD2. The information on structure and function of endosomes that contain DRD2 are still lacking. We suppose they represent late endosomes and hope that future studies aiming to define their origin will help us understand the whole cycle of molecular events, from DRD2 cell membrane expression and internalization to their storage and reuse.

dopamine receptors; expression pattern; immunocytochemistry; immunofluorescence; neuroblastoma cell line; SH-SY5Y; receptor internalization

Nagrada za najbolji poster