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...

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Interaction between two alphabeta half-receptors within the (alphabeta)(2) holoreceptor complex is required for insulin binding with high affinity and for insulin-triggered changes of size and shape. To understand the underlying structure-function relationship, two truncated receptor constructs have been characterized. Reduction in the Stokes radius and increase in the sedimentation coefficient, which are characteristic for wild-type receptors, were entirely lacking for the recombinant human insulin receptor (HIR) ectodomain (HIR-ED). Stokes radii of about 5.8 nm and sedimentation coefficients of 10.2 S were found for both insulin-bound and free HIR-EDs. However, attaching the membrane anchors to the ectodomain, as with the recombinant membrane-anchored ectodomain (HIR-MAED) construct, was sufficient to restore not only high-affinity hormone binding but also the marked insulin-inducible alterations in hydrodynamic properties. The Stokes radii of HIR-MAED complexes, as assessed by non-denaturing PAGE, decreased upon insulin binding from 9.5 nm to 7.9 nm. In parallel, the sedimentation coefficient was increased from 9.0 S to 9.8 S. CD and fluorescence spectroscopy of HIR-MAED revealed only minor insulin-induced changes in the secondary structure. Similarity with wild-type receptors has also been demonstrated by the differential insertion of insulin-bound and free HIR-MAED complexes into artificial bilayer membranes of Triton X-114. The results are consistent with a model of receptor function that ensures a global insulin-triggered reorientation of subdomains within the ectodomain moieties while the secondary structure is essentially retained. For the rearrangement of such subdomains, the transmembrane anchors confer essential structural constraints on the receptor ectodomain.

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