title: Determination of affinity and activity of ligands at the human neuropeptide Y Y4 receptor by flow cytometry and aequorin luminescence.
abstract: Fluorescence-labeled neuropeptide Y (NPY) has been used in flow cytometric binding assays for the determination of affinity constants of NPY Y1, Y2, and Y5 receptor ligands. Because the binding of fluorescent NPY is insufficient for competition studies at the human Y4 receptor (hY4R), we replaced Glu-4 in hPP with Lys for the derivatization with cyanine-5. Because cy5-[K(4)]hPP has high affinity (Kd 5.6 nM) to the hY4R, it was used as a probe in a flow cytometric binding assay. Specific binding of cy5-[K(4)]hPP to hY4R was visualized by confocal microscopy. The hY(4)R, the chimeric G protein G(qi5) and mitochondrially targeted apoaequorin were stably coexpressed in CHO cells. Aequorin luminescence was quantified in a microplate reader and by a CCD camera. By application of these methods 3-cyclohexyl-N-[(3-1H-imidazol-4-ylpropylamino)(imino)methyl]propanamide (UR-AK49) was discovered as the first nonpeptidic Y4R antagonist (pKi 4.17), a lead to be optimized in terms of potency and selectivity.
authors: Institut für Pharmazie, Universität Regensburg, Regensburg, Germany.
authors: Ralf
authors: R
authors: Ziemek
authors: Erich
authors: E
authors: Schneider
authors: Anja
authors: A
authors: Kraus
authors: Chiara
authors: C
authors: Cabrele
authors: Annette G
authors: AG
authors: Beck-Sickinger
authors: Günther
authors: G
authors: Bernhardt
authors: Armin
authors: A
authors: Buschauer
mesh_terms: D000331:Aequorin / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D049449:Luminescence; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D011485:Protein Binding; D017476:Receptors, Neuropeptide Y / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
chemical_list: D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; D000331:Aequorin; C109491:neuropeptide Y4 receptor