BODIPY®-conjugated neuropeptide Y ligands:: new fluorescent tools to tag Y1, Y2, Y4 and Y5 receptor subtypes

1 N-terminal labelled fluorescent BODIPY (R)-NPY peptide analogues were tested in Y1, Y2, Y4 and Y-5 receptor-binding assays performed in rat brain membrane preparations and HEK293 cells expressing the rat Y1, Y2, Y4 and Y5 receptors. 2 BODIPY (R) TMR/FL-[Leu(31), Pro(34)] NPY/ PYY were able to compete for specific [ 125][ Leu31, Pro(34)]PYY-binding sites with an affinity similar to that observed for the native peptide at the Y1 (K-i = 1 - 6 nM), Y-2 ( K-i > 1000 nM), Y-4 ( K-i = 10 nM) and Y-5 (K-i = 1-4 nM) receptor subtypes. 3 BODIPY (R) FL-PYY(3 - 36) was able to compete for specific Y-2 (K-i = 10 nM) and Y5 (Ki = 30 nM) binding sites, but had almost no affinity in Y-1 and Y-4 assays. 4 BODIPY (R) FL-hPP was able to compete with high affinity (K-i; 1 and 15 nM) only in Y4 and Y5 receptor-binding assays. 5 BODIPY (R) TMR-[cPP(1 - 7), NPY(19 - 23), Ala(31), Aib(32), Gln(34)] hPP and BODIPY (R) TMR-[hPP(1-17), Ala(31), Aib(32)]NPY were potent competitors only on specific Y-5-binding sites (K-i = 0.1 - 0.6 nM). 6 As expected, these fluorescent peptides inhibited forskolin-induced cAMP accumulation, demonstrating that they retained their agonist properties. 7 When tested in confocal microscopy imaging, fluorescent Y-1 and Y-5 agonists internalized in a time-dependent manner in Y-1 and Y-5 transfected cells, respectively. 8 These results demonstrate that BODIPY (R)-conjugated NPY analogues retain their selectivity, affinity and agonist properties for the Y-1, Y-2, Y-4 and Y-5 receptor subtypes, respectively. Thus, they represent novel tools to study and visualize NPY receptors in living cells.