The GTPase RalA regulates different steps of the secretory process in pancreatic beta-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 beta-cells and analyzed the impact on different steps of the insulin-secretory process.


RESULTS
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
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 beta-cell by coordinating the execution of different events in the secretory pathway.
Ljubicic
Sanda
S
Department of Cell Biology and Morphology, University of Lausanne, Lausanne, Switzerland.
Bezzi
Paola
P
Vitale
Nicolas
N
Regazzi
Romano
R
D000818:Animals; D005786:Gene Expression Regulation; D020868:Gene Silencing; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology; D007700:Kinetics; D051379:Mice; D008957:Models, Genetic; D010739:Phospholipase D / Q000378:metabolism; D020033:Protein Isoforms; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D020725:ral GTP-Binding Proteins / Q000378:metabolism*
D020033:Protein Isoforms; D010739:Phospholipase D; C118120:Rala protein, mouse; C492197:Rala protein, rat; D020725:ral GTP-Binding Proteins