Identification of a potent inverse agonist at a constitutively active mutant of human P2Y12 receptor
Identification of a potent inverse agonist at a constitutively active mutant of human P2Y12 receptor
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DOI：
10.1124/mol.105.014654
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发表时间：
2006-01-01
影响因子：
3.6
通讯作者：
Kunapuli, SP
中科院分区：
文献类型：
--
作者：
Ding, ZG;Kim, S;Kunapuli, SP
人血小板表达两种P2 Y受体：G（q）-偶联的P2 Y 1和G（i）-偶联的P2 Y（12）。P2 Y（1）和P2 Y（12）都是人血小板上的ADP受体，并且对于ADP诱导的血小板聚集是必需的，血小板聚集在血栓形成和止血中起关键作用。在天然或重组系统中已经描述了许多组成型活性的G蛋白偶联受体，但在P2 Y受体中，迄今为止，还没有报道组成型活性。为了鉴定人血小板ADP受体的G蛋白偶联结构域，我们构建了一个嵌合的血凝素标记的人P2 Y（12）受体，其C端被人P2 Y（1）受体的相应部分取代，并在中国仓鼠卵巢K1细胞中稳定表达。有趣的是，嵌合P2 Y（12）突变体表现出高水平的组成型活性，如在不存在激动剂的情况下降低的cAMP水平所证明的。G（i）抑制剂百日咳毒素可显著抑制嵌合P2 Y（12）突变体的组成性激活。组成型活性P2 Y（12）突变体保留了对2-甲硫基-ADP的正常反应，EC 50为0.15 +/- 0.04 nM。组成型活性P2 Y（12）突变体引起Akt磷酸化，而加入百日咳毒素可消除Akt磷酸化。几种P2 Y（12）拮抗剂的药理学评价显示（E）-N-[1-[7-（己基氨基）-5（丙硫基）-3-H-1，2，3-三唑并-[4，5-d]-嘧啶-3-基]-1，5，6-三脱氧-β-D-核糖-庚-5-烯呋喃脲酰基]-L-天冬氨酸（AR-C78511）作为有效的P2 Y（12）反向激动剂和5 '-腺苷酸，N-[2-（甲硫基）乙基]-2-[（3，3，3-三氟丙基）硫基]-，单酐与（二氯亚甲基）双[膦酸]（AR-C69931 MX）作为中性拮抗剂。总之，这是第一个稳定表达人血小板P2 Y（12）受体组成型活性突变体的细胞系和鉴定有效的反向激动剂的报告。
Human platelets express two P2Y receptors: G(q)-coupled P2Y 1, and G(i)-coupled P2Y(12). Both P2Y(1) and P2Y(12) are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems, but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of the human platelet ADP receptor, we constructed a chimeric hemagglutinin-tagged human P2Y(12) receptor with its C terminus replaced by the corresponding part of human P2Y(1) receptor and stably expressed it in Chinese hamster ovary-K1 cells. It is interesting that the chimeric P2Y(12) mutant exhibited a high level of constitutive activity, as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y(12) mutant was dramatically inhibited by pertussis toxin, a G(i) inhibitor. The constitutively active P2Y(12) mutant retained normal responses to 2-methylthio-ADP, with an EC50 of 0.15 +/- 0.04 nM. The constitutively active P2Y(12) mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y(12) antagonists revealed (E)-N-[1-[7-(hexylamino)-5(propylthio)-3-H-1,2,3-triazolo-[4,5-d]-pyrimidin-3-yl]-1,5,6trideoxy-beta-D-ribo-hept-5-enofuranuronoyl]-L-aspartic acid (AR-C78511) as a potent P2Y(12) inverse agonist and 5'-adenylic acid, N-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl) thio]-, monoanhydride with (dichloromethylene)bis[phosphonic acid] (AR-C69931MX) as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y(12) receptor and the identification of potent inverse agonist.