Involvement of basic amino acid residues in transmembrane regions 6 and 7 in agonist and antagonist recognition of the human platelet P2Y12-receptor
Involvement of basic amino acid residues in transmembrane regions 6 and 7 in agonist and antagonist recognition of the human platelet P2Y12-receptor
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DOI：
10.1016/j.bcp.2008.08.029
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发表时间：
2008-11-15
影响因子：
5.8
通讯作者：
von Kuegelgen, Ivar
中科院分区：
文献类型：
--
作者：
Hoffmann, Kristina;Sixel, Uta;von Kuegelgen, Ivar
P2 Y（12）受体在ADP诱导的血小板聚集中起重要作用。在本研究中，我们寻找参与人P2 Y（12）-受体配体识别的氨基酸残基。野生型或突变型受体在1321 N1星形细胞瘤细胞和中国仓鼠卵巢（CHO）细胞中表达。构建体的细胞表达没有重大差异。异丙肾上腺素（星形细胞瘤细胞）或毛喉素（CHO细胞）增加细胞CAMP生产和CAMP反应元件（CRE）依赖性荧光素酶表达。在表达野生型受体、R256 K或S101 A突变体构建体的细胞中，2-甲硫基-ADP抑制诱导的CAMP产生，IC 50浓度约为0.3 nM。在表达R256 A构建体的细胞中，IC 50浓度总计为25 nM。在表达H253 A/R256 A、Y259 D和K280 A构建体的细胞中，2-甲硫基-ADP不能影响细胞cAMP的产生。此外，在表达Y259 D和K280 A构建体的细胞中，2-甲硫基-ADP也没有改变毛喉素诱导的CRE依赖性荧光素酶表达，仅引起血清反应元件依赖性荧光素酶表达的小幅增加。拮抗剂坎格雷洛对野生型受体和R256 A构建体具有相似的效力（野生型受体的表观pK（B）值：9.2）。相反，活性蓝-2对R256 A构建体具有较低的效力（在野生型受体处的表观pK（B）值：7.6）。总之，数据表明Arg 256、Tyr 259和可能的H253（跨膜区TM 6）以及Lys 280（TM 7）参与人P2 Y12受体的功能。Arg 256似乎在识别核苷酸激动剂和非核苷酸拮抗剂活性蓝-2中起作用，但在识别核苷酸拮抗剂坎格雷洛中没有作用。(C)2008年爱思唯尔公司All rights reserved.
The P2Y(12)-receptor plays a prominent role in ADP-induced platelet aggregation. In the present study, we searched for amino acid residues involved in ligand recognition of the human P2Y(12)-receptor. Wild-type or mutated receptors were expressed in 1321N1 astrocytoma cells and Chinese hamster ovary (CHO) cells. There were no major differences in cellular expression of the constructs. Cellular CAMP production and CAMP response element (CRE)-dependent luciferase expression was increased by isoproterenol (astrocytoma cells) or forskolin (CHO cells). In cells expressing wild-type receptors, R256K or S101A mutant constructs, 2-methylthio-ADP inhibited the induced CAMP production with IC50 concentrations of about 0.3 nM. In cells expressing R256A constructs, the IC50 concentration amounted to 25 nM. In cells expressing H253A/R256A, Y259D and K280A constructs, 2-methylthio-ADP failed to affect the cellular CAMP production. Moreover, in cells expressing Y259D and K280A constructs, 2-methylthio-ADP did also not change the forskolin-induced CRE-dependent luciferase expression and caused only small increases in the serum response element-dependent luciferase expression. The antagonist cangrelor had similar potencies at wildtype receptors and R256A constructs (apparent pK(b)-value at wild-type receptors: 9.2). In contrast, reactive blue-2 had a lower potency at the R256A construct (apparent pK(B)-value at wild-type receptors: 7.6). In summary, the data indicate the involvement of Arg256, Tyr259 and, possibly, H253 (transmembrane region TM6) as well as Lys280 (TM7) in the function of the human P2Y12-receptor. Arg256 appears to play a role in the recognition of nucleotide agonists and the non-nucleotide antagonist reactive blue-2, but no role in the recognition of the nucleotide antagonist cangrelor. (C) 2008 Elsevier Inc. All rights reserved.