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Epac2 signaling at the β-cell plasma membrane
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. (Anders Tengholm)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Secretion of appropriate amounts of insulin from pancreatic β-cells is crucial for glucose homeostasis. The β-cells release insulin in response to glucose and other nutrients, hormones and neurotransmitters, which trigger intracellular signaling cascades, that result in exocytotic fusion of insulin-containing vesicles with the plasma membrane. Increases of the intracellular concentration of calcium ions ([Ca2+]i) trigger exocytosis, whereas the messenger cyclic adenosine monophosphate (cAMP) amplifies various steps of the secretion process. The protein Epac2 mediates some effects of cAMP, but little is known about its regulation in β-cells. In this study, the spatio-temporal dynamics of Epac2 was investigated in insulin-secreting MIN6-cells and primary β-cells using various cell signaling biosensors and live-cell fluorescence microscopy approaches. Increases in the cAMP concentration triggered translocation of Epac2 from the cytoplasm to the plasma membrane. Oscillations of cAMP induced by glucose and the insulin-releasing hormone GLP-1 were associated with cyclic translocation of Epac2. Analyses of Epac2 mutants showed that the high-affinity cyclic nucleotide-binding domain and Ras-association domains were crucial for the translocation, whereas neither the DEP domain, nor the low-affinity cAMP-binding domain were required for membrane binding. However, the latter domain targeted Epac2 to insulin granules at the plasma membrane, which promoted their priming for exocytosis. Depolarization-induced elevations of [Ca2+]i also stimulated Epac2 translocation, but the effects were complex and in the presence of high cAMP concentrations, [Ca2+]i increases often reduced membrane binding. The stimulatory effect of Ca2+ was mediated by increased Ras activity, while the inhibitory effect reflected reduced concentrations of the membrane phospholipid PtdIns(4,5)P2. Anti-diabetic drugs of the sulfonylurea class, suggested to directly activate Epac2, induced translocation indirectly by depolarizing β-cells to increase [Ca2+]i. Epac2 is an activator of Rap GTPases, and its translocation increased Rap activity at the plasma membrane. It is concluded that the subcellular localization of Epac2 is controlled by a complex interplay between cAMP, Ca2+ and PtdIns(4,5)P2 and that the protein controls insulin release by binding to the exocytosis machinery. These results provide new insights into the regulation of β-cell function and may facilitate the development of new anti-diabetic drugs that amplify insulin secretion.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 51 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1227
Keyword [en]
β-cell, insulin secretion, cAMP, Ca2+, Epac, Rap, exocytosis, sulfonylurea
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-284638ISBN: 978-91-554-9580-0 (print)OAI: oai:DiVA.org:uu-284638DiVA: diva2:920771
Public defence
2016-06-08, A1:107a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-05-17 Created: 2016-04-19 Last updated: 2016-06-01
List of papers
1. Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic beta Cells
Open this publication in new window or tab >>Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic beta Cells
2013 (English)In: Science Signaling, ISSN 1945-0877, E-ISSN 1937-9145, Vol. 6, no 273, ra29- p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-201253 (URN)10.1126/scisignal.2003932 (DOI)000318350100002 ()
Available from: 2013-06-10 Created: 2013-06-10 Last updated: 2017-12-06Bibliographically approved
2. Two-step membrane recruitment of Epac2 primes secretory granules for exocytosis
Open this publication in new window or tab >>Two-step membrane recruitment of Epac2 primes secretory granules for exocytosis
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-284568 (URN)
Available from: 2016-04-19 Created: 2016-04-18 Last updated: 2016-06-01
3. Localization of Epac2 to the plasma membrane is controlled by an interplay between cAMP, Ca2+ and PtdIns(4,5)P2
Open this publication in new window or tab >>Localization of Epac2 to the plasma membrane is controlled by an interplay between cAMP, Ca2+ and PtdIns(4,5)P2
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-284580 (URN)
Available from: 2016-04-19 Created: 2016-04-18 Last updated: 2016-06-01
4. Sulfonylureas trigger Ca2+-dependent Epac2 recruitment and Rap activation at the plasma membrane in β-cells
Open this publication in new window or tab >>Sulfonylureas trigger Ca2+-dependent Epac2 recruitment and Rap activation at the plasma membrane in β-cells
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-284582 (URN)
Available from: 2016-04-19 Created: 2016-04-18 Last updated: 2016-06-01

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