Characterisation of Neuropeptide Y Receptor Subtypes by Synthetic NPY Analogues and by Anti-receptor Antibodies

Christophe P Eckard,Annette G Beck-Sickinger,Heike A Wieland,Chiara Cabrele

doi:10.3390/60500448

Christophe P Eckard, Annette G Beck-Sickinger + Show 2 more

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https://doi.org/10.3390/60500448

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Abstract

Neuropeptide Y (NPY), a 36-mer neuromodulator, binds to the receptors Y1, Y2, Y4 and Y5 with nanomolar affinity. They all belong to the rhodopsin-like G-protein coupled, seven transmembrane helix spanning receptors. In this study, Ala-substituted and centrally truncated NPY analogues were compared with respect to affinity to the Y-receptors. Furthermore, antibodies against the second (E2) and the third (E3) extracellular loop of NPY Y1-, Y2- and Y5-receptor subtypes were raised and affinity to intact cells was tested by immunofluorescence assays. Both methods were applied in order to receive subtype selective tools and to characterise ligand binding. The analogues [A13]-pNPY and [A27]-pNPY showed subtype selectivity for the Y2-receptor. Sera against the E2 loop of the Y1-receptor and against the E2 loop of the Y2-receptor were subtype selective. Two antibodies against the Y5 E2 and E3 loop recognised the Y5- and Y2-receptor subtypes. In combination, these sera are able to distinguish between the Y1-, Y2-, and Y5-receptor subtypes. The analogues and antibodies represent valuable tools to distinguish NPY receptors on membranes and intact cells.

Highlights

Neuropeptide Y (NPY) is a 36 amino acid peptide hormone that belongs to the pancreatic polypeptide hormone family [1]
In this study we describe synthetic NPY analogues tested in binding assays against the Y-receptors and we compare the binding of antibodies raised against the second and the third extracellular loop of NPY Y1, Y2- and Y5-receptor subtypes to intact cells in immunofluorescence assays
All peptides were designed as amino acid mono-substituted full length or centrally truncated analogues of porcine neuropeptide Y (pNPY) and human pancreatic polypeptide (hPP) and were obtained by multiple automatic solid phase peptide synthesis using the Fmoc strategy

Summary

Introduction

Neuropeptide Y (NPY) is a 36 amino acid peptide hormone that belongs to the pancreatic polypeptide hormone family [1]. It is widely distributed both peripherally and centrally. Various other biological effects have been attributed to NPY, e.g., profound effects on secretion of luteinising hormone as well as on growth hormone and insulin release [3,4,5]. These observations suggest the important role of NPY in the pathophysiology of obesity and diabetes. Five distinct NPY receptor subtypes have been cloned and pharmacologically characterised [16]. The broad physiological relevance of NPY gives reason for an increasing interest in NPY as a new target in drug discovery

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