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Dynamic control of Epac2 localization by cAMP and Ca2+-mediated activation of Ras
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Epac2, a cAMP-regulated guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2, is an important mediator of a variety of cAMP-regulated cellular processes, including insulin secretion from pancreatic β-cells. Epac2 has been suggested to associate with the plasma membrane by interacting with active Ras (Ras-GTP), but the dynamics and regulation of membrane binding is unknown. Using real-time confocal and total internal reflection fluorescence microscopy we demonstrate that cAMP-elevating agents cause rapid translocation of GFP-tagged Epac2 from the cytoplasm to the plasma membrane in insulin-secreting MIN6 β-cells. Glucose concentrations that stimulate insulin secretion often triggered oscillatory translocation of GFP-Epac2 following oscillations of the sub-membrane concentrations of cAMP and Ca2+ ([cAMP]pm and [Ca2+]pm). The translocation was suppressed after inhibition of adenylyl cyclases or removal of extracellular Ca2+. GFP-Epac2 translocation by rise of [Ca2+]pm required concomitant elevation of [cAMP]pm and cAMP-induced translocation was enhanced by moderate [Ca2+]pm elevations. However the effect of Ca2+ was dual since translocation was inhibited by high [Ca2+]pm spikes. Epac2 mutants lacking the cAMP-binding or Ras-association domains were unable to translocate and localized constitutively to the plasma membrane and cytoplasm, respectively. Ras activity monitored with a fluorescent Ras-GTP binding reporter was tightly correlated with the translocation of Epac2. It is concluded that Epac2 localization is dynamically controlled by cAMP as well as by Ca2+-mediated activation of Ras, and that reversible translocation of Epac2 between the cytoplasm and plasma membrane requires both Ras-association and cAMP-binding domains. Spatio-temporal control of Epac2 in β-cells has implications for the understanding of its involvement in insulin secretion kinetics by Rap GTPases and other downstream effectors at the plasma membrane.

Keyword [en]
cAMP, Ca2+, Epac2, Ras, beta-cell, evanescent wave microscopy, glucose
National Category
Cell Biology
Research subject
Medical Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-113684OAI: oai:DiVA.org:uu-113684DiVA: diva2:291655
Available from: 2010-02-02 Created: 2010-02-02 Last updated: 2010-02-03
In thesis
1. Oscillatory Signaling and Insulin Secretion from Single ß-cells
Open this publication in new window or tab >>Oscillatory Signaling and Insulin Secretion from Single ß-cells
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

cAMP and Ca2+ are key regulators of exocytosis in many cells, including insulin-secreting pancreatic β-cells. Glucose-stimulated insulin secretion from β-cells is pulsatile and driven by oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i), but little is known about the kinetics of cAMP signaling and the mechanisms of cAMP action. Evanescent wave microscopy and fluorescent translocation biosensors were used to monitor plasma membrane-related signaling events in single MIN6-cells and primary mouse β-cells. Glucose stimulation of insulin secretion resulted in pronounced oscillations of the membrane phospholipid PIP3 caused by autocrine activation of insulin receptors. Glucose also triggered oscillations of the sub-plasma membrane cAMP concentration ([cAMP]pm). These oscillations were preceded and enhanced by elevations of [Ca2+]i, but conditions raising cytoplasmic ATP triggered [cAMP]pm elevations without accompanying changes in [Ca2+]i. The [cAMP]pm oscillations were also synchronized with PIP3 oscillations and both signals were suppressed after inhibition of adenylyl cyclases. Protein kinase A (PKA) was important for promoting concomitant initial elevations of [cAMP]pm and [Ca2+]i, and PKA inhibitors diminished the PIP3 response when applied before glucose stimulation, but did not affect already manifested PIP3 oscillations. The glucose-induced PIP3 oscillations were markedly suppressed in cells treated with siRNA against the cAMP-dependent guanine nucleotide exchange factor Epac2. Pharmacological activation of Epac restored PIP3 responses after adenylyl cyclase or PKA inhibition. Glucose and other cAMP-elevating stimuli induced redistribution of fluorescence-tagged Epac2 from the cytoplasm to the plasma membrane. This translocation was modulated by [Ca2+]i and depended on intact cyclic nucleotide-binding and Ras-association domains. In conclusion, glucose generates cAMP oscillations in β-cells via a concerted action of Ca2+ and metabolically generated ATP. The oscillations are important for the magnitude and kinetics of insulin secretion. While both protein kinase A and Epac is required for initiation of insulin secretion the cAMP-dependence of established pulsatility is mediated by Epac2.

Place, publisher, year, edition, pages
Uppsala: A U U, 2010. 71 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 520
Keyword
cAMP, Ca2+, oscillations, beta-cell, insulin secretion, evanescent wave microscopy, PIP3, PKA, Epac
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-113686 (URN)978-91-554-7718-9 (ISBN)
Public defence
2010-03-19, B21, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-02-25 Created: 2010-02-02 Last updated: 2010-02-25Bibliographically approved

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