A Y2 receptor mimetic aptamer directed against neuropeptide Y.
Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that exerts its activity by at least five different receptor subtypes that belong to the family of G-protein-coupled receptors. We isolated an aptamer directed against NPY from a nuclease-resistant RNA library. Mapping experiments with N-terminally, C-terminally, and centrally truncated analogues of NPY revealed that the aptamer recognizes the C terminus of NPY. Individual replacement of the four arginine residues at positions 19, 25, 33, and 35 by l-alanine showed that arginine 33 is essential for binding. The aptamer does not recognize pancreatic polypeptide, a highly homologous Y4 receptor-specific peptide of the gut. Furthermore, the affinity of the aptamer to the Y5 receptor-selective agonist [Ala(31),Aib(32)]NPY and the Y1/Y5 receptor-binding peptide [Leu(31),Pro(34)]NPY was considerably reduced, whereas Y2 receptor-specific NPY mutants were bound well by the aptamer. Accordingly, the NPY epitope was recognized by the Y2 receptor, and the aptamer was highly similar. This Y2 receptor mimicking effect was further confirmed by competition binding studies. Whereas the aptamer competed with the Y2 receptor for binding of [(3)H]NPY with high affinity, a low affinity displacement of [(3)H]NPY was observed at the Y1 and the Y5 receptors. Consequently, competition at the Y2 receptor occurred with a considerably lower K(i) value compared with the Y1 and Y5 receptors. These results indicate that the aptamer mimics the binding of NPY to the Y2 receptor more closely than to the Y1 and Y5 receptors.
Proske
Daniela
D
Kekulé Institut für Organische Chemie und Biochemie, Universität Bonn, Germany.
Höfliger
Martin
M
Söll
Richard M
RM
Beck-Sickinger
Annette G
AG
Famulok
Michael
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000235:genetics; D017476:Receptors, Neuropeptide Y / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; C095889:neuropeptide Y2 receptor; D012313:RNA