Monitoring of the internalization of neuropeptide Y on neuroblastoma cell line SK-N-MC.
Neuropeptide Y (NPY) is an important neuromodulator in the central and peripheral nervous system. The peptide acts through different NPY receptor subtypes (Y1-Y5, y6) that belong to the family of G protein-coupled receptors. In general, cellular responses to prolonged exposure to agonists of G protein-coupled receptors are attenuated, often through internalization of the receptors and their bound ligands. In this study, a fluorescent labeled NPY derivative was synthesized and characterized to investigate the internalization of NPY in the human neuroblastoma cell line SK-N-MC. Internalization was proven by binding experiments and subsequent acidic washing as well as by direct visualization by means of confocal laser scanning microscopy. Approximately 20-30% of the fluorescent labeled NPY and a tritium-marked NPY were resistant to acid removal of cell surface-bound ligands indicating internalization. Extracellular fluorescent labeled NPY was found to be distributed heterogeneously in a clustered pattern, which suggests that the ligand-receptor complex is collected in pits and caveolae followed by endocytosis.
Fabry
M
M
German Wool Research Institute at the University of Technology Aachen, Germany. fabry@dwi.rwth-aachen.de
Langer
M
M
Rothen-Rutishauser
B
B
Wunderli-Allenspach
H
H
Höcker
H
H
Beck-Sickinger
A G
AG
D000595:Amino Acid Sequence; D004705:Endocytosis; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D009478:Neuropeptide Y / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay; D017476:Receptors, Neuropeptide Y / Q000378:metabolism; D014407:Tumor Cells, Cultured
D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y