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

N‐terminal labelled fluorescent BODIPY®‐NPY peptide analogues were tested in Y 1 , Y 2 , Y 4 and Y 5 receptor‐binding assays performed in rat brain membrane preparations and HEK293 cells expressing the rat Y 1 , Y 2 , Y 4 and Y 5 receptors. BODIPY®TMR/FL‐[Leu 31 , Pro 34 ]NPY/PYY were able to compete for specific [ 125 ][Leu 31 , Pro 34 ]PYY‐binding sites with an affinity similar to that observed for the native peptide at the Y 1 ( K i =1–6 n M ), Y 2 ( K i >1000 n M ), Y 4 ( K i =10 n M ) and Y 5 ( K i =1–4 n M ) receptor subtypes. BODIPY®FL‐PYY(3–36) was able to compete for specific Y 2 ( K i =10 n M ) and Y 5 ( K i =30 n M ) binding sites, but had almost no affinity in Y 1 and Y 4 assays. BODIPY®FL‐hPP was able to compete with high affinity ( K i ; 1 and 15 n M ) only in Y 4 and Y 5 receptor‐binding assays. BODIPY®TMR‐[cPP(1–7), NPY(19–23), Ala 31 , Aib 32 , Gln 34 ]hPP and BODIPY®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 n M ). As expected, these fluorescent peptides inhibited forskolin‐induced cAMP accumulation, demonstrating that they retained their agonist properties. 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. These results demonstrate that BODIPY®‐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.British Journal of Pharmacology (2005) 146 , 1069–1081. doi: 10.1038/sj.bjp.0706425