title: Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y2 receptor ligands.
abstract: With respect to the discovery and characterization of neuropeptide Y(2) receptor ligands as pharmacological tools or potential drugs, fluorescence- and luminescence-based assays were developed to determine both the affinity and the activity of receptor agonists and antagonists. A flow cytometric binding assay is described for the hY(2) receptor stably expressed in CHO cells using cy5-labeled porcine neuropeptide Y and compared with a radioligand binding assay. Binding of the fluorescent ligand was visualized by confocal microscopy. Stable co-transfection with the chimeric G protein Gq(i5) enabled the establishment of a spectrofluorimetric fura-2 and a flow cytometric fluo-4 calcium assay. Further stable expression of apoaequorin targeted to the mitochondria allowed the establishment of an aequorin assay which could be performed in the 96-well format. The shape of the concentration-response curves of porcine neuropeptide Y in the presence of the Y(2)-selective receptor antagonist BIIE0246, characteristic of either competitive or insurmountable antagonism, depended on the period of incubation with the cells. Functional data of Y(2) receptor agonists and antagonists determined in the fluorescence- and luminescence-based assays were in good agreement.
authors.affiliation: University of Regensburg, Institute of Pharmacy, Universitätsstr. 31, D-93040 Regensburg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Ziemek
authors.forename: Albert
authors.initials: A
authors.lastname: Brennauer
authors.forename: Erich
authors.initials: E
authors.lastname: Schneider
authors.forename: Chiara
authors.initials: C
authors.lastname: Cabrele
authors.forename: Annette G
authors.initials: AG
authors.lastname: Beck-Sickinger
authors.forename: Günther
authors.initials: G
authors.lastname: Bernhardt
authors.forename: Armin
authors.initials: A
authors.lastname: Buschauer
mesh_terms: D000331:Aequorin / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001059:Apoproteins / Q000187:drug effects / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D001552:Benzazepines / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D016466:CHO Cells; D020013:Calcium Signaling / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000187:drug effects / Q000378:metabolism; D008024:Ligands; D018613:Microscopy, Confocal / Q000379:methods*; D009478:Neuropeptide Y / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D019894:Peptide YY / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011869:Radioligand Assay; D017476:Receptors, Neuropeptide Y / Q000187:drug effects / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013552:Swine; D014162:Transfection
chemical_list: D001059:Apoproteins; D001552:Benzazepines; D005456:Fluorescent Dyes; D008024:Ligands; D009478:Neuropeptide Y; D010446:Peptide Fragments; D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C060014:apoaequorin; C062924:neuropeptide Y (13-36); C095889:neuropeptide Y2 receptor; D019894:Peptide YY; D000331:Aequorin; D001120:Arginine; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C403256:BIIE 0246