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. 2011 Feb 18;286(7):5319-27.
doi: 10.1074/jbc.M110.176933. Epub 2010 Nov 8.

Demonstration of angiotensin II-induced Ras activation in the trans-Golgi network and endoplasmic reticulum using bioluminescence resonance energy transfer-based biosensors

András Balla  1 , László Sándor Erdélyi, Eszter Soltész-Katona, Tamas Balla, Péter Várnai, László Hunyady
Affiliations

Demonstration of angiotensin II-induced Ras activation in the trans-Golgi network and endoplasmic reticulum using bioluminescence resonance energy transfer-based biosensors

András Balla et al. J Biol Chem. .

Abstract

Previous studies have demonstrated that molecules of the Ras signaling pathway are present in intracellular compartments, including early endosomes, the endoplasmic reticulum (ER), and the Golgi, and suggested that mitogens can regulate Ras activity in these endomembranes. In this study, we investigated the effect of angiotensin II (AngII) on intracellular Ras activity in living HEK293 cells expressing angiotensin type 1 receptors (AT(1)-Rs) using newly developed bioluminescence resonance energy transfer biosensors. To investigate the subcellular localization of AngII-induced Ras activation, we targeted our probes to various intracellular compartments, such as the trans-Golgi network (TGN), the ER, and early endosomes. Using these biosensors, we detected AngII-induced Ras activation in the TGN and ER, but not in early endosomes. In cells expressing a cytoplasmic tail deletion AT(1)-R mutant, the AngII-induced response was enhanced, suggesting that receptor internalization and β-arrestin binding are not required for AngII-induced Ras activation in endomembranes. Although we were able to demonstrate EGF-induced Ras activation in the plasma membrane and TGN, but not in other endomembranes, AG1478, an EGF receptor inhibitor, did not affect the AngII-induced response, suggesting that the latter is independent of EGF receptor transactivation. AngII was unable to stimulate Ras activity in the studied compartments in cells expressing a G protein coupling-deficient AT(1)-R mutant ((125)DRY(127) to (125)AAY(127)). These data suggest that AngII can stimulate Ras activity in the TGN and ER with a G protein-dependent mechanism, which does not require β-arrestin-mediated signaling, receptor internalization, and EGF receptor transactivation.

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Figures

FIGURE 1.
FIGURE 1.
Ras activation upon AngII and EGF stimulation in HEK293 cells. A, intramolecular RasBRET-tK; B, intramolecular MP-RasBRET; C, intermolecular MP-RasBRET. Upper rows, schematic representation of the arrangement of the RasBRET probes. Middle rows, BRET assay of plasma membrane-targeted RasBRET probes. HEK293 cells were transfected with the plasmids of the indicated probes and with the AT1A-R, and after 24 h, the serum-starved cells were exposed to 50 ng/ml EGF (blue traces), 100 nm AngII (red traces), or vehicle (dotted lines) at the indicated time points. The BRET records are the average of at least three independent experiments. The means ± S.E. are shown (n = 3). Lower rows, the representative confocal micrographs show the localization and cellular distribution of the indicated probes in HEK293 cells. YFP fluorescence was detected using a Zeiss LSM 510 confocal microscope. Stim, stimulation. Scale bars = 10 μm.
FIGURE 2.
FIGURE 2.
Validation of the BRET measurement approach for agonist-stimulated Ras activation by mutated MP-RasBRET probe pairs. HEK293 cells were transfected with intermolecular RasBRET pairs. The wild-type (●), active G12V (■), and inactive S17N (♦) MP-RasBRET probes are shown. HEK293 cells were transfected with the plasmids of the indicated probes and with the AT1A-R, and after 24 h, the serum-starved cells were exposed to 50 ng/ml EGF (blue trace), 100 nm AngII (red trace), or vehicle (dashed line) at the indicated time points. The BRET records are an average of at least three independent experiments. The means ± S.E. are shown (n = 3). Stim, stimulation.
FIGURE 3.
FIGURE 3.
Ras activation upon agonist stimulation of different AT1-R mutants. HEK293 cells were cotransfected with intermolecular MP-RasBRET and the wild-type AT1A-R, AT1A-RΔ319, or AT1A-R-DRY/AAY plasmid. The serum-starved cells were exposed to 100 nm AngII (open triangles, dashed line for the wild-type AT1A-R, open rectangles, dotted line for AT1A-RΔ319, and open circles for AT1A-R-DRY/AAY) or vehicle (dashed line) at the indicated time points. The BRET records are an average of at least three independent experiments. The means ± S.E. are shown (n = 3). Stim, stimulated.
FIGURE 4.
FIGURE 4.
Ras activation in intracellular organelles. HEK293 cells were cotransfected with the indicated intermolecular RasBRET probes along with either the wild-type AT1A-R (left panels) or AT1A-RΔ319 (middle panels) plasmid. HEK293 cells were exposed to 50 ng/ml EGF (filled symbols), 100 nm AngII (open symbols), or vehicle (dashed lines) at the indicated time points. The BRET records are an average of at least three independent experiments, each performed in triplicate. The means ± S.E. are shown (n = 3). A–C, Ras activation in the TGN, the ER, and endosomes, respectively. The representative confocal micrographs (right panels) show the localization and cellular distribution of the various probes in HEK293 cells that were transfected with the plasmids of the indicated probes. YFP fluorescence was detected using a Zeiss LSM 510 confocal microscope. Because the endosome- and ER-targeting sequences are located at the C terminus of RBD-luciferase, the micrographs for endosomes and the ER show the cellular distribution of the intramolecular versions of RasBRET-Sac1 and RasBRET-FYVE, respectively. Stim, stimulated.
FIGURE 5.
FIGURE 5.
Mechanism of Ras activation upon AngII stimulation is independent of EGF receptor transactivation in HEK293 cells. HEK293 cells were transfected with the AT1A-R and with the plasmid pair of the intermolecular MP-RasBRET (A) or TGN-RasBRET (B), and after 24 h, the serum-starved cells were pretreated for 30 min prior to BRET measurement either with vehicle (left panels) or with 1 μm AG1478 (right panels). After the pretreatment, the cells were exposed to 50 ng/ml EGF (filled symbols), 100 nm AngII (open symbols, dotted lines), or vehicle (dashed traces) at the indicated time points. The BRET records are an average of at least three independent experiments, each performed in triplicate. The means ± S.E. are shown (n = 3). Stim, stimulated.
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