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 peri pheral nervous system. The peptide acts through different NPY receptor subt ypes (Y1-Y5, y6) that belong to the family of G protein-coupled receptors. In general, cellular responses to prolonged exposure to agonists of G prote in-coupled receptors are attenuated, often through internalization of the r eceptors and their bound ligands. In this study, a fluorescent labeled NPY derivative was synthesized and characterized to investigate the internaliza tion of NPY in the human neuroblastoma cell line SK-N-MC. Internalization w as proven by binding experiments and subsequent acidic washing as well as b y direct visualization by means of confocal laser scanning microscopy. Appr oximately 20-30% of the fluorescent labeled NPY and a tritium-marked NPY we re resistant to acid removal of cell surface-bound ligands indicating inter nalization. Extracellular fluorescent labeled NPY was found to be distribut ed heterogeneously in a clustered pattern, which suggests that the ligand-r eceptor complex is collected in pits and caveolae followed by endocytosis.