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

Background

RalA 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 Findings

We 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/Significance

Taken 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.

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