Spatial Control of Epac2 Activity by cAMP and Ca ²⁺ -Mediated Activation of Ras in Pancreatic β Cells

Abstract

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 β 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 Ca(2+) 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 Ca(2+)-dependent oscillations in Ras activity synchronized with Epac2 translocation in single β 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 Ca(2+)-mediated activation of Ras. These results help to explain how oscillating signals can produce pulses of insulin release from pancreatic β cells.

Impaired cAMP Generation Contributes to Defective Glucose-Stimulated Insulin Secretion After Long-term Exposure to Palmitate

“…The viability of cells within intact mouse islets was assessed by incubating islets for 15 min in experimental buffer supplemented with 10 mg/mL propidium iodide (Sigma) and 2 mmol/L calcein acetoxymethyl ester (Life Technologies) followed by imaging with a confocal microscope (described by Idevall-Hagren et al [28]). The area of propidium iodide stained nuclei in relation to the calceinstained viable cells was calculated with ImageJ (29).…”

Endocrinology of the Single Cell

“…7,21,22,40 Oscillatory insulin secretion is well established, and the subcellular events generating this pattern became a bit more clear with the report in single cells that glucose triggers coordinated oscillations of the sub-plasma membrane Ca 21 and cAMP concentrations, synergizing to generate pulsatile insulin secretion (Figure 20.1B). 22 Submembrane oscillations of cAMP and Ca 21 have also been demonstrated in response to GLP-1 in single β-cells (Figure 20.1C).…”

“…Using real-time confocal and TIRF microscopy in single cells, Idevall-Hagren and colleagues report that an increase in submembrane cAMP recruits Epac translocation to the plasma membrane, and this translocation is cyclic in the presence of elevated glucose (Figure 20.2A). 7 Further, the Epac2 oscillations are accompanied by oscillations of Rap GTPase activity in the submembrane area, positioning Rap1 at potential sites of exocytosis. In cells in which Epac2 is knocked down by siRNA or in cells expressing a dominant-negative Rap1b mutant (Figure 20.2B), the magnitude of the pulsatile insulin secretion indicator in response to either glucose stimulation or K 1 stimulation in single cells is significantly reduced.…”

“…To some extent, this problem can be bypassed with the use of current tools, which allow for single-cell manipulation and real-time data sampling from intact tissue, for example single β-cells within pancreatic islets or single neurons within hypothalamic slices. 7,8 Another challenge that is the byproduct of current innovative single-cell technology can be a mass of multidimensional data generated that, in some cases, may require mathematical modeling and new analytical tools. 9 Finally, the combination of single-cell tools required for experimental manipulation, data acquisition, and multiplex analysis may be costly and require skills that come with a steep learning curve.…”

“…The methodologies range from the simple visualization of individual cells to aid electrophysiological studies to laser scanning microscopy and the use of total internal reflection fluorescence microscopy (TIRFM) to study, for example, the mechanisms involved in secretory granule dynamics 12 or the subplasma membrane oscillations in concentration of Ca 21 and cAMP ( Figure 20.1). 7 Fluorescence microscopy also is an essential tool in optogenetics studies and in the examination of molecular interactions using Fö rster resonance energy transfer (FRET; also fluorescence resonance energy transfer) and bioluminescence resonance energy transfer (BRET). 11 Light-activated molecules can provide precise temporal and spatial control and, recently, optogenetics has provided a wealth of tools for studying single cell dynamics.…”