Isoform-specific antagonists of exchange proteins directly activated by cAMP
Isoform-specific antagonists of exchange proteins directly activated by cAMP
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DOI：
10.1073/pnas.1210209109
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发表时间：
2012-11-06
影响因子：
11.1
通讯作者：
Cheng, Xiaodong
中科院分区：
文献类型：
--
作者：
Tsalkova, Tamara;Mei, Fang C.;Cheng, Xiaodong
哺乳动物细胞中cAMP的主要生理作用是通过两种普遍表达的细胞内cAMP受体，即蛋白激酶A （PKA）和cAMP直接激活的交换蛋白（EPAC），以及某些组织中的环核苷酸门控离子通道来转导的。尽管有大量的PKA抑制剂可用，但尽管进行了广泛的研究，尚无报道的epac特异性拮抗剂。在这里，我们报告了对EPAC2亚型特异性的非环核苷酸EPAC拮抗剂的鉴定和表征。这些EAPC2特异性拮抗剂分别为ESI-05和ESI-07，它们能高效抑制由EAPC2介导的Rap1激活，而不抑制EPAC1。此外，ESI-05和ESI-07能够抑制camp介导的EPAC2的激活，但不能抑制EPAC1和PKA，这是通过使用epac和PKA为基础的FRET报告器或使用Rap1-GTP下拉试验在活细胞中监测到的。氘交换质谱分析进一步揭示epac2特异性抑制剂通过一种独特的机制发挥其异构体选择性，通过结合先前描述的变构位点：两个cAMP结合域的界面，这在EPAC1异构体中不存在。异构体特异性EPAC药理学探针是非常需要的，将是解剖EPAC蛋白的生物学功能及其在各种疾病状态中的作用的有价值的工具。
The major physiological effects of cAMP in mammalian cells are transduced by two ubiquitously expressed intracellular cAMP receptors, protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC), as well as cyclic nucleotide-gated ion channels in certain tissues. Although a large number of PKA inhibitors are available, there are no reported EPAC-specific antagonists, despite extensive research efforts. Here we report the identification and characterization of noncyclic nucleotide EPAC antagonists that are exclusively specific for the EPAC2 isoform. These EAPC2-specific antagonists, designated as ESI-05 and ESI-07, inhibit Rap1 activation mediated by EAPC2, but not EPAC1, with high potency in vitro. Moreover, ESI-05 and ESI-07 are capable of suppressing the cAMP-mediated activation of EPAC2, but not EPAC1 and PKA, as monitored in living cells through the use of EPAC-and PKA-based FRET reporters, or by the use of Rap1-GTP pull-down assays. Deuterium exchange mass spectroscopy analysis further reveals that EPAC2-specific inhibitors exert their isoform selectivity through a unique mechanism by binding to a previously undescribed allosteric site: the interface of the two cAMP binding domains, which is not present in the EPAC1 isoform. Isoform-specific EPAC pharmacological probes are highly desired and will be valuable tools for dissecting the biological functions of EPAC proteins and their roles in various disease states.