title: BODIPY-conjugated neuropeptide Y ligands: new fluorescent tools to tag Y1, Y2, Y4 and Y5 receptor subtypes.
abstract: N-terminal labelled fluorescent BODIPY-NPY peptide analogues were tested in Y1, Y2, Y4 and Y5 receptor-binding assays performed in rat brain membrane preparations and HEK293 cells expressing the rat Y1, Y2, Y4 and Y5 receptors. BODIPY TMR/FL-[Leu31, Pro34]NPY/PYY were able to compete for specific [125][Leu31, Pro34]PYY-binding sites with an affinity similar to that observed for the native peptide at the Y1 (Ki=1-6 nM), Y2 (Ki>1000 nM), Y4 (Ki=10 nM) and Y5 (Ki=1-4 nM) receptor subtypes. BODIPY FL-PYY(3-36) was able to compete for specific Y2 (Ki=10 nM) and Y5 (Ki=30 nM) binding sites, but had almost no affinity in Y1 and Y4 assays. BODIPY FL-hPP was able to compete with high affinity (Ki; 1 and 15 nM) only in Y4 and Y5 receptor-binding assays. BODIPY TMR-[cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP and BODIPY TMR-[hPP(1-17), Ala31, Aib32]NPY were potent competitors only on specific Y5-binding sites (Ki=0.1-0.6 nM). As expected, these fluorescent peptides inhibited forskolin-induced cAMP accumulation, demonstrating that they retained their agonist properties. When tested in confocal microscopy imaging, fluorescent Y1 and Y5 agonists internalized in a time-dependent manner in Y1 and Y5 transfected cells, respectively. These results demonstrate that BODIPY-conjugated NPY analogues retain their selectivity, affinity and agonist properties for the Y1, Y2, Y4 and Y5 receptor subtypes, respectively. Thus, they represent novel tools to study and visualize NPY receptors in living cells.
authors.affiliation: Department of Psychiatry, Douglas Hospital Research Centre, McGill University, Montreal (Verdun), QC, Canada H4H 1R3.
authors.forename: Yvan
authors.initials: Y
authors.lastname: Dumont
authors.forename: Pierrette
authors.initials: P
authors.lastname: Gaudreau
authors.forename: Manuela
authors.initials: M
authors.lastname: Mazzuferi
authors.forename: Daniel
authors.initials: D
authors.lastname: Langlois
authors.forename: Jean-Guy
authors.initials: JG
authors.lastname: Chabot
authors.forename: Alain
authors.initials: A
authors.lastname: Fournier
authors.forename: Michele
authors.initials: M
authors.lastname: Simonato
authors.forename: Rémi
authors.initials: R
authors.lastname: Quirion
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001896:Boron Compounds; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure; D002460:Cell Line / Q000378:metabolism / Q000648:ultrastructure; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008297:Male; D009478:Neuropeptide Y / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D010191:Pancreatic Polypeptide / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D019894:Peptide YY / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017476:Receptors, Neuropeptide Y / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D008024:Ligands; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; C097212:neuropeptide Y-Y1 receptor; C095889:neuropeptide Y2 receptor; C100568:neuropeptide Y5 receptor; D019894:Peptide YY; D005576:Colforsin; C109491:neuropeptide Y4 receptor; D010191:Pancreatic Polypeptide; D000242:Cyclic AMP