Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic β Cells
Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic β Cells
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DOI：
10.1126/scisignal.2003932
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发表时间：
2013-04-30
影响因子：
7.3
通讯作者：
Tengholm, Anders
中科院分区：
文献类型：
--
作者：
Idevall-Hagren, Olof;Jakobsson, Ida;Tengholm, Anders
cAMP（腺苷3′，5′-单磷酸腺苷）激活的鸟嘌呤核苷酸交换因子（GEF） Epac2是多种细胞类型中cAMP依赖过程的重要介质。我们使用实时共聚焦和全内反射荧光显微镜研究了Epac2的时空调控，Epac2是鸟苷三磷酸酶（GTPase） Rap的GEF。我们证明，在胰岛素分泌β细胞中，cAMP浓度的增加触发了Epac2从细胞质向质膜的易位。葡萄糖诱导的膜下cAMP浓度振荡与Epac2的循环易位有关，这种易位可以通过细胞质Ca2+浓度的增加而放大。对Epac2突变体的分析发现，高亲和力的camp结合和Ras关联结构域对易位至关重要。Ras显性阴性突变体的表达减少了Epac2易位，并且在单个β细胞中，Ras活性的Ca2+依赖振荡与Epac2易位同步。Epac2的循环易位伴随着Rap GTPase在质膜上的活性振荡，并且失活Rap1B突变体的表达降低了胰岛素分泌。因此，Epac2的定位是由cAMP和Ca2+介导的Ras激活动态控制的。这些结果有助于解释振荡信号如何产生胰腺b细胞释放胰岛素的脉冲。
The cAMP (adenosine 3',5'-monophosphate)-activated guanine nucleotide exchange factor (GEF) Epac2 is an important mediator of cAMP-dependent processes in multiple cell types. We used real-time confocal and total internal reflection fluorescence microscopy to examine the spatiotemporal regulation of Epac2, which is a GEF for the guanosine triphosphatase (GTPase) Rap. We demonstrated that increases in the concentration of cAMP triggered the translocation of Epac2 from the cytoplasm to the plasma membrane in insulin-secreting beta cells. Glucose-induced oscillations of the submembrane concentration of cAMP were associated with cyclic translocation of Epac2, and this translocation could be amplified by increases in the cytoplasmic Ca2+ concentration. Analyses of Epac2 mutants identified the high-affinity cAMP-binding and the Ras association domains as crucial for the translocation. Expression of a dominant-negative Ras mutant reduced Epac2 translocation, and Ca2+-dependent oscillations in Ras activity synchronized with Epac2 translocation in single beta cells. The cyclic translocation of Epac2 was accompanied by oscillations of Rap GTPase activity at the plasma membrane, and expression of an inactive Rap1B mutant decreased insulin secretion. Thus, Epac2 localization is dynamically controlled by cAMP as well as by Ca2+-mediated activation of Ras. These results help to explain how oscillating signals can produce pulses of insulin release from pancreatic b cells.