The GTPase RalA Regulates Different Steps of the Secretory Process in Pancreatic β-Cells

BackgroundRalA and RalB are multifuntional GTPases involved in a variety of cellular processes including proliferation, oncogenic transformation and membrane trafficking. Here we investigated the mechanisms leading to activation of Ral proteins in pancreatic β-cells and analyzed the impact on different steps of the insulin-secretory process.
Methodology/Principal FindingsWe found that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-1E cells by RNA interference led to a decrease in secretagogue-induced insulin release. Real-time measurements by fluorescence resonance energy transfer revealed that RalA activation in response to secretagogues occurs within 3–5 min and reaches a plateau after 10–15 min. The activation of the GTPase is triggered by increases in intracellular Ca2+ and cAMP and is prevented by the L-type voltage-gated Ca2+ channel blocker Nifedipine and by the protein kinase A inhibitor H89. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane detected by Total Internal Reflection Fluorescence microscopy and with a strong impairment in Phospholipase D1 activation in response to secretagogues. RalA was found to be activated by RalGDS and to be severely hampered upon silencing of this GDP/GTP exchange factor. Accordingly, INS-1E cells lacking RalGDS displayed a reduction in hormone secretion induced by secretagogues and in the number of insulin-containing granules docked at the plasma membrane.
Conclusions/SignificanceTaken together, our data indicate that RalA activation elicited by the exchange factor RalGDS in response to a rise in intracellular Ca2+ and cAMP controls hormone release from pancreatic β-cell by coordinating the execution of different events in the secretory pathway.

研究背景：RalA与RalB为多功能GTP酶（GTPase），参与细胞增殖、致癌转化及膜运输（membrane trafficking）等多种细胞生物学过程。本研究旨在探究胰腺β细胞中Ral蛋白的激活机制，并分析其对胰岛素分泌过程不同环节的影响。

研究方法与主要结果：我们发现，RalA是胰岛及分泌胰岛素的细胞系中表达占优的同工型。通过RNA干扰（RNA interference, RNAi）技术沉默INS-1E细胞中的该GTP酶，可使促分泌剂诱导的胰岛素释放量显著降低。采用荧光共振能量转移（fluorescence resonance energy transfer, FRET）进行实时检测发现，RalA在促分泌剂刺激下的激活发生于3~5分钟内，并在10~15分钟后达到平台期。该GTP酶的激活由细胞内Ca²⁺与cAMP水平升高所触发，可被L型电压门控钙通道阻滞剂硝苯地平（Nifedipine）以及蛋白激酶A抑制剂H89所阻断。通过全内反射荧光显微镜（Total Internal Reflection Fluorescence microscopy, TIRFM）检测发现，缺失RalA的细胞中，锚定在质膜上的分泌颗粒数量减少，且促分泌剂诱导的磷脂酶D1（Phospholipase D1, PLD1）激活过程受到严重损害。研究还发现，RalA可被RalGDS激活，而沉默该GDP/GTP交换因子会显著抑制RalA的激活。与之相符的是，缺失RalGDS的INS-1E细胞中，促分泌剂诱导的激素分泌量以及锚定在质膜上的含胰岛素颗粒数量均出现降低。

研究结论与意义：综合来看，本研究数据表明，在细胞内Ca²⁺与cAMP水平升高时，交换因子RalGDS介导的RalA激活，可通过协调分泌通路中的不同事件，调控胰腺β细胞的激素释放过程。