Gehlert Donald R, Schober Douglas A, Morin Michelle, Berglund Magnus M
Lilly Neuroscience, Lilly Research Laboratories, Eli Lilly and Co., Lilly Corporate Center, Indianapolis, IN 46285, United States.
Biochem Pharmacol. 2007 Dec 3;74(11):1652-64. doi: 10.1016/j.bcp.2007.08.017. Epub 2007 Aug 19.
Centrally administered neuropeptide Y (NPY) produces anxiolytic and orexigenic effects by interacting with Y1 and Y5 receptors that are colocalized in many brain regions. Therefore, we tested the hypothesis that co-expression of Y1 and Y5 receptors results in heterodimerization, altered pharmacological properties and altered desensitization. To accomplish this, the carboxyl-termini of Y1 and Y5 receptors were fused with Renilla luciferase and green fluorescent protein and the proximity of the tagged receptors assessed using bioluminescent resonance energy transfer. Under basal conditions, cotransfection of tagged Y1 receptor and Y5 produced a substantial dimerization signal that was unaffected by the endogenous, nonselective agonists, NPY and peptide YY (PYY). Selective Y5 agonists produced an increase in the dimerization signal while Y5 antagonists also produced a slight but significant increase. In the absence of agonists, selective antagonists decreased dimerization. In functional studies, Y5 agonists produced a greater inhibition of adenylyl cyclase activity in Y1/Y5 cells than cells expressing Y5 alone while NPY and PYY exhibited no difference. With PYY stimulation, the Y1 antagonist became inactive and the Y5 antagonist exhibited uncompetitive kinetics in the Y1/Y5 cell line. In confocal microscopy studies, Y1/Y5 co-expression resulted in increased Y5 signaling following PYY stimulation. Addition of both Y1 and Y5 receptor antagonists was required to significantly decrease PYY-induced internalization. Therefore, Y1/Y5 co-expression results in heterodimerization, altered agonist and antagonist responses and reduced internalization rate. These results may account for the complex pharmacology observed when assessing the responses to NPY and analogs in vivo.
中枢给予的神经肽Y（NPY）通过与在许多脑区共定位的Y1和Y5受体相互作用产生抗焦虑和促食欲作用。因此，我们检验了Y1和Y5受体共表达导致异源二聚化、药理学特性改变和脱敏改变的假说。为实现这一点，将Y1和Y5受体的羧基末端与海肾荧光素酶和绿色荧光蛋白融合，并使用生物发光共振能量转移评估标记受体的接近程度。在基础条件下，标记的Y1受体和Y5共转染产生了大量的二聚化信号，该信号不受内源性非选择性激动剂NPY和肽YY（PYY）的影响。选择性Y5激动剂使二聚化信号增加，而Y5拮抗剂也产生轻微但显著的增加。在没有激动剂的情况下，选择性拮抗剂降低二聚化。在功能研究中，Y5激动剂在Y1/Y5细胞中对腺苷酸环化酶活性的抑制作用比单独表达Y5的细胞更大，而NPY和PYY则无差异。在PYY刺激下，Y1拮抗剂失活，Y5拮抗剂在Y1/Y5细胞系中表现出非竞争性动力学。在共聚焦显微镜研究中，Y1/Y5共表达导致PYY刺激后Y信号增加。需要同时添加Y1和Y5受体拮抗剂才能显著降低PYY诱导的内化。因此，Y1/Y5共表达导致异源二聚化、激动剂和拮抗剂反应改变以及内化率降低。这些结果可能解释了在体内评估对NPY及其类似物的反应时观察到的复杂药理学现象。
