1. 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 receptor. 2. 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 to 6 nM), Y2 (Ki > 1000 nM), Y4 (Ki = 10 nM) and Y5 (Ki = 1 to 4 nM) receptor subtypes. 3. 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. 4. BODIPY®FL-hPP was able to compete with high affinity (Ki; 1 and 15 nM) only in Y4 and Y5 receptor binding assays. 5. BODIPY®TMR-[cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP and BODIPY®TMR-[hPP(1-17), Ala31, Aib32]NPY were potent competitors for specific Y5 sites (Ki = 0.1 to 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 Y1 and Y5 agonists internalized in a time-dependent manner in transfected Y1 and Y5 transfected cells, respectively. 8. These results demonstrate that BODIPY®-conjugated NPY analogues retain their selectivity, affinity and agonist properties for the Y1, Y2, Y4 and Y5 receptor subtype, respectively. Thus, they represent novel tools to study and visualize NPY receptors in living cells.
BODIPY (R)-conjugated neuropeptide Y ligands: new fluorescent tools to tag Y1, Y2, Y4 and Y5 receptor subtypes
MAZZUFERI, Manuela;SIMONATO, Michele;
2005
Abstract
1. 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 receptor. 2. 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 to 6 nM), Y2 (Ki > 1000 nM), Y4 (Ki = 10 nM) and Y5 (Ki = 1 to 4 nM) receptor subtypes. 3. 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. 4. BODIPY®FL-hPP was able to compete with high affinity (Ki; 1 and 15 nM) only in Y4 and Y5 receptor binding assays. 5. BODIPY®TMR-[cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP and BODIPY®TMR-[hPP(1-17), Ala31, Aib32]NPY were potent competitors for specific Y5 sites (Ki = 0.1 to 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 Y1 and Y5 agonists internalized in a time-dependent manner in transfected Y1 and Y5 transfected cells, respectively. 8. These results demonstrate that BODIPY®-conjugated NPY analogues retain their selectivity, affinity and agonist properties for the Y1, Y2, Y4 and Y5 receptor subtype, respectively. Thus, they represent novel tools to study and visualize NPY receptors in living cells.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
