Functional characterization of human neuropeptide Y receptor subtype five specific antagonists using a luciferase reporter gene assay.
Neuropeptide Y (NPY) has several receptors; one of them, the neuropeptide Y5 receptor (NPY5) seems involved in feeding behavior in mammals. Although this particular receptor has been extensively studied in the literature, the difficulties encountered to obtain a stable cell line expressing this recombinant receptor have impaired the development of tools necessary to establish its molecular pharmacology. We thus established a method for the functional study of new ligands. It is based upon the cotransfection in human melatonin receptor 1 (MT1)-overexpressing HEK293 cells of three plasmids encoding melanocortin receptor (MC5), neuropeptide Y5 receptor (NPY5) and a cyclic AMP response element-controlled luciferase. Once challenged with alphaMSH, the MC5 receptor activates the cyclic AMP response, through the coupling protein subunit G(s). In contrast, NPY5 agonists, through the NPY5 receptor which is negatively coupled to the same pathway, counteract the alphaMSH-mediated effect on cyclic AMP level. Using appropriate controls, this method can pinpoint compounds with antagonistic activity. Simple and straightforward, this system permits reproducible measurements of agonist or antagonist effects in the presence of neuropeptide Y, the natural agonist. This method has the advantage over already existing methods and beyond its apparent complexity, to enhance the cyclic AMP concentration at a 'physiological' level, by opposition to a forskolin-induced adenylate cyclase activation. Finally, to further validate this assay, we showed results from (1) a series of natural peptidic agonists that permitted the standardization and (2) a series of potent nonpeptidic antagonists (affinity >10(-9) M) that form a new class of active NPY5 receptor antagonists.
Beauverger
Philippe
P
Division de Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, 125 Chemin de Ronde, 78 290 Croissy-sur-Seine, France.
Rodriguez
Marianne
M
Nicolas
Jean-Paul
JP
Audinot
Valérie
V
Lamamy
Véronique
V
Dromaint
Sandra
S
Nagel
Nadine
N
Macia
Christelle
C
Léopold
Odile
O
Galizzi
Jean-Pierre
JP
Caignard
Daniel-Henri
DH
Aldana
Ignacio
I
Monge
Antonio
A
Chomarat
Pascale
P
Boutin
Jean A
JA
D001681:Biological Assay; D017930:Genes, Reporter; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000235:genetics*; D009478:Neuropeptide Y / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D017476:Receptors, Neuropeptide Y / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D008024:Ligands; D009478:Neuropeptide Y; D010455:Peptides; D017476:Receptors, Neuropeptide Y; D011994:Recombinant Proteins; C100568:neuropeptide Y5 receptor; D008156:Luciferases