Monitoring of the internalization of neuropeptide Y on neuroblastoma cell line SK‐N‐MC

Abstract

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 surfacebound 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. Keywords: neuropeptide Y (NPY); internalization; neuroblastoma cells; fluorescent labeling.Neuropeptide Y (NPY) is a neurotransmitter consisting of 36 amino acids that belongs to the family of pancreatic polypeptides. These polypeptides are important modulators of the central and peripheral nervous system and NPY is one of the most abundant neuropeptides in the brain. NPY acts peripherally as a vasoconstrictor and modulates the activity of further neurotransmitters. Centrally, NPY is involved in the stimulating of food intake, memory retention and anxiolysis. So far, five receptor subtypes (Y1, Y2, Y4, Y5, y6) were identified that bind NPY with nanomolar affinity [1]. They all belong to the large family of G protein-coupled receptors (GPCR) with their typical seven-transmembrane helix structure [2].Approximately 80% of all bioactive molecules, especially hormones and neurotransmitters, act through the interaction of GPCR that transduce extracellular signals to the interior of cells. Signaling through these receptors, however, is rapidly desensitized primarily as a consequence of receptor phosphorylation, but internalization and receptor downregulation have also been shown to occur [3]. Many GPCR are known to internalize after agonist binding, however, the conditions for this mechanism and its importance for the desensitization and resensitization as well as for the fate of receptor and ligand within the cells are different and often unclear.Regulatory mechanisms of signal attenuation, including the removal of the ligands from the extracellular space by enzymatic degradation, are important to ensure the ability of the cells to react to the agonists. Degradation of the peptide and desensitization of the NPY signal have been reported [4,5]. Recently, investigations showed that CHO cells expressing the Y4 receptor were resistant to agonist-promoted desensitization and internalization [6].In the present study, we e...

Towards improved receptor targeting: anterograde transport, internalization and postendocytic trafficking of neuropeptide Y receptors

“…The hY 1 receptor was found to be rapidly internalized upon agonist stimulation by different groups (Fabry et al , 2000 ;Gicquiaux et al , 2002 ;Bohme et al , 2008 ;Ouedraogo et al , 2008 ;Lindner et al , 2009 ;Kilpatrick et al , 2010 ;Lecat et al , 2011 ;Lundell et al , 2011 ), consistent with the results that were obtained for its orthologs in the rat (Holliday and Cox , 2003 ;Pheng et al , 2003 ;Holliday et al , 2005 ;Kilpatrick et al , 2012 ) or guinea pig (Parker et al , 2001(Parker et al , , 2002b. Several studies on the internalization mechanism revealed that the Y 1 receptor undergoes clathrin-dependent endocytosis in an Arr3-dependent fashion and is transported back to the cell membrane, involving direct and indirect recycling mechanisms (Gicquiaux et al , 2002 ;Berglund et al , Pheng et al , 2003b ;Holliday et al , 2005 ;Ouedraogo et al , 2008 ;Kilpatrick et al , 2010 ;Lecat et al , 2011 ).…”

Rapid Internalization and Recycling of the Human Neuropeptide Y Y1 Receptor

“…Recycling is generally thought to occur after the agonist has been washed out, but there is evidence for GPCRs recycling back to the plasma membrane both in the presence and absence of agonist (55,56). In our experiments, we could not determine whether the agonist hNPY is recycled with the receptor back to the cell surface or whether it is degraded in lysosomes (23,24).…”

“…However, neither the underlying molecular mechanisms nor the intracellular trafficking pathways involved are well known. Recent studies indicate that endogenous Y 1 receptors in SK-N-MC neuroblastoma (23) and guinea pig Y 1 but not Y 2 receptors expressed in CHO cells (24) are internalized. On the other hand, in pharmacological studies, hNPY Y 4 receptors expressed in CHO cells were resistant to desensitization and internalization (25), although in radioligand studies, rat Y 4 receptors expressed in CHO cells were found to be internalized (24).…”

The Use of Bioluminescence Resonance Energy Transfer 2 to Study Neuropeptide Y Receptor Agonist-Induced β-Arrestin 2 Interaction

“…All four NPY family receptors can couple to G i but have also been found to evoke an increase in intracellular Ca 2ϩ (Berglund et al, 2003), contradicting the implication that Y1 and possibly Y5 are class B receptors. In addition, several groups have reported that the Y1 receptor internalizes into endosomes, leading to a rapid recycling of the receptor to the cell surface (Fabry et al, 2000;Gicquiaux et al, 2002), a typical feature of class A receptors.…”

“…This may ultimately lead to internalization of the receptor. The Y1 and Y4 receptors have been shown to internalize rapidly upon agonist binding (Fabry et al, 2000;Parker et al, 2001;Gicquiaux et al, 2002;Parker et al, 2002), whereas it has been suggested that Y2 does not internalize or does so very slowly (Parker et al, 2001;Gicquiaux et al, 2002).…”