Dumont Yvan, Gaudreau Pierrette, Mazzuferi Manuela, Langlois Daniel, Chabot Jean-Guy, Fournier Alain, Simonato Michele, Quirion Rémi
Department of Psychiatry, Douglas Hospital Research Centre, McGill University, Montreal (Verdun), QC, Canada H4H 1R3.
Br J Pharmacol. 2005 Dec;146(8):1069-81. doi: 10.1038/sj.bjp.0706425.
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.
在大鼠脑膜制剂和表达大鼠Y1、Y2、Y4和Y5受体的HEK293细胞中进行的Y1、Y2、Y4和Y5受体结合试验中,对N端标记的荧光BODIPY-NPY肽类似物进行了测试。BODIPY TMR/FL-[Leu31, Pro34]NPY/PYY能够与特异性[125I][Leu31, Pro34]PYY结合位点竞争,其亲和力与天然肽在Y1(Ki=1-6 nM)、Y2(Ki>1000 nM)、Y4(Ki=10 nM)和Y5(Ki=1-4 nM)受体亚型上观察到的相似。BODIPY FL-PYY(3-36)能够与特异性Y2(Ki=10 nM)和Y5(Ki=30 nM)结合位点竞争,但在Y1和Y4试验中几乎没有亲和力。BODIPY FL-hPP仅在Y4和Y5受体结合试验中能够以高亲和力(Ki分别为1和15 nM)竞争。BODIPY TMR-[cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP和BODIPY TMR-[hPP(1-17), Ala31, Aib32]NPY仅在特异性Y5结合位点上是有效的竞争者(Ki=0.1-0.6 nM)。正如预期的那样,这些荧光肽抑制了福司可林诱导的cAMP积累,表明它们保留了激动剂特性。在共聚焦显微镜成像中进行测试时,荧光Y1和Y5激动剂分别在Y1和Y5转染细胞中以时间依赖性方式内化。这些结果表明,BODIPY缀合的NPY类似物分别对Y1、Y2、Y4和Y5受体亚型保留了其选择性、亲和力和激动剂特性。因此,它们代表了研究和可视化活细胞中NPY受体的新型工具。