Demonstration of Angiotensin II-induced Ras Activation in the trans-Golgi Network and Endoplasmic Reticulum Using Bioluminescence Resonance Energy Transfer-based Biosensors^*

Materials

Molecular biology enzymes were obtained from Fermentas (Burlington, Canada), Stratagene (La Jolla, CA), and Invitrogen. Cell culture dishes and plates for BRET measurements were purchased from Geiner Bio-One GmbH (Kremsmunster, Austria). Lipofectamine 2000 and coelenterazine h were from Invitrogen. Unless stated otherwise, all other chemicals and reagents were purchased from Sigma. HEK293 cells were from American Type Culture Collection (Manassas, VA).

Molecular Biology

For the construction of our intramolecular BRET probes, we followed the design of the Raichu (Ras and interacting protein chimeric unit) FRET probes from the Matsuda laboratory (26, 27). The cDNA parts of the constructs were generated by amplification either from a respective expressed sequence tag clone (German Resource Center for Genome Research (RZPD), Berlin, Germany) or from H295R human cell line cDNA by PCR. For the intramolecular BRET probes, the order of the domains was similar to that in the Raichu probes (26). We used the pEYFP-C1 plasmid backbone (Clontech, Mountain View, CA), and the fluorescent acceptor protein (YFP) was followed by H-Ras, the Ras-binding domain (RBD) of Raf-1, and luciferase (see Fig. 1 for schematic representation). We have previously shown that the Raf-1 RBD (amino acids 51–131) is able to recognize activated Ras but is not capable of being recruited to the plasma membrane (28), which makes this RBD suitable for utilizing in targeted biosensors. We used Renilla luciferase as the energy donor by subcloning the codon-humanized Renilla luciferase from pRluc-N2 (PerkinElmer Life Sciences). The intermolecular BRET probes were derived from their respective intramolecular probes by splitting them between Ras and the RBD of Raf-1 by adding suitable start and stop codon sequences. Targeting was achieved by placing a targeting signal at either the N- or C-terminal end of the probe. We used several previously described targeting motifs to target our BRET probes (29). Targeting to the plasma membrane was achieved using various distinct tags. We fused CAAX motifs from Ras proteins C-terminally as described (26, 30). Briefly, the tK tag (K-Ras CAAX targeting motif, tail K-Ras), KMSKDGKKKKKKSKTKCVIM, consists of the membrane-targeting CAAX motif and hypervariable regions of K-Ras (GenBank accession number NM_004985). The tHL tag (tail H-Ras long, KLNPPDESGPGCMSCKCVLS) consists of the membrane-targeting CAAX motif and hypervariable regions of H-Ras (accession number NM_005343), whereas the tHS tag (tail H-Ras short, CMSCKCVLS) consists of the membrane-targeting CAAX motif but without the hypervariable regions of H-Ras. The MP (MyrPalm) and PP (PalmPalm) tags targeting to plasma membrane rafts were based on the design of Zacharias et al. (31), where the MP tag is the N-terminal amino acid sequence (MGCIKSKRKDNLNDDE) from Lyn kinase (accession number NM_002350), and the PP tag is the N-terminal amino acid sequence (MLCCMRRTKQ) from GAP43 (accession number NM_002045) (31). Targeting to the Golgi surface was achieved by fusing the full-length trans-Golgi network protein 2 (also known as TGN38; accession number NM_006464) (32), and targeting to the ER surface was achieved by adding segment 233–250 of the yeast Ubc6 protein (accession number NM_001178991) or the C-terminal segment 521–587 of the Sac1 phosphoinositide phosphatase (accession number NM_014016) (29, 33). Endosomal targeting was done by fusing the probes C-terminally with the FYVE domain (segment 1253–1411) of EEA1 (early endosome antigen 1; accession number NM_3566). Mutagenesis was performed using standard mutagenesis conditions. After verifying the mutations with dideoxy sequencing, the mutated fragment was exchanged between the wild-type and mutated portions with suitable restriction sites to avoid the generation of unwanted mutations outside the sequenced regions. pcDNA3.1-AT_1A-R, pcDNA3.1-AT_1A-RΔ319 (with Tyr³¹⁹ substituted with a stop codon), and pcDNA3.1-DRY/AAY-AT_1A-R (with the highly conserved ¹²⁵DRY¹²⁷ sequence mutated to ¹²⁵AAY¹²⁷) were described previously (34–36).

Cell Culture and Transfection

The experiments were performed on the HEK293 cell line. The cells were cultured in DMEM with penicillin/streptomycin (Invitrogen) and 10% heat-inactivated FBS in 5% CO₂ at 37 °C. The cells were cultured in plastic dishes, trypsinized prior to transfection, and transiently transfected using Lipofectamine 2000 (Invitrogen), and plated on polylysine-pretreated white 96-well plates at a density of 1 × 10⁵ cells/well for BRET measurements. The DNA amount was 0.2–0.4 μg/well; the amount of Lipofectamine 2000 was 0.5 μl/well.

BRET Measurements

We monitored the molecular interactions of either two parts of a biosensor (intramolecular probes) or two proteins (intermolecular probes) by BRET measurements. The measurements were performed after 24 h of transfection on white 96-well plates. Before BRET measurements, the HEK293 cells were switched to serum-free medium (DMEM with 0.1% BSA) for several hours (4–6 h) to render the cells quiescent. The medium of the cells was changed prior to measurements to a modified Krebs-Ringer buffer containing 120 mm NaCl, 4.7 mm KCl, 1.8 mm CaCl₂, 0.7 mm MgSO₄, 10 mm glucose, and 10 mm NaHEPES (pH 7.4), and the BRET measurements were performed at 37 °C. The BRET measurements were started after the addition of the cell-permeable substrate coelenterazine at a final concentration of 5 μm, and the counts were recorded using a Berthold Mithras LB 940 multilabel reader, which allows for the detection of signals using filters at 485- and 530-nm wavelengths. The detection time was 0.25–0.5 s. The BRET ratios were calculated as the 530/485 nm ratio. The measurements were done in triplicate. The BRET records are the average of at least three independent experiments. BRET ratios were base line-corrected to the vehicle curve using GraphPad Prism software. The approximate BRET ratio of the YFP-deficient intramolecular RasBRET-tK probe was ∼0.8 (data not shown).

Confocal Microscopy

The localization and distribution of the targeted probes were analyzed using a Zeiss LSM 510 confocal laser scanning microscope in living cells plated on polylysine-pretreated glass coverslips (3 × 10⁵ cells/35-mm dish).

Generation and Optimization of RasBRET Probes to Measure Ras Activation upon AngII Stimulation in HEK293 cells

To study Ras activation after AngII or EGF stimulation in intracellular compartments, we first established a BRET-based Ras activity measurement using newly developed probes. For the detection of Ras activation, HEK293 cells were chosen for our studies because this cell line is broadly used for angiotensin receptor studies (20, 23, 37). Moreover, this cell type has endogenous EGF receptors, and Ras activation upon EGF treatment could be detected using BRET measurements. Stimulation of endogenous EGF receptors by EGF yielded a robust and prolonged response in Ras activation as assessed in the plasma membrane by the intramolecular RasBRET-tK probe (see details under “Experimental Procedures”) (Fig. 1A), which was designed as the BRET version of the Raichu-Ras fusion protein (26). The probes indicate when the respective Ras becomes loaded with GTP because the RBD of Raf-1 increases its affinity dramatically for Ras upon GTP loading. This results in conformational changes that bring the energy acceptor close to the donor, as was shown by the developers of the corresponding FRET probes (38). Compared with EGF treatment, Ras activation upon AngII stimulation resulted in a transient and smaller response in HEK293 cells (Fig. 1A).

To optimize the probe, tags were used to target it to different plasma membrane microdomains, including non-raft domains (tK), caveolae/rafts (tHS and tHL) (39), and plasma membrane raft domains (MP and PP) (31). The localization and distribution of the targeted probes in HEK293 cells were verified by confocal microscopy (Fig. 1, lower panels). Among these targeting sequences, the MP-targeted version of the intramolecular probe yielded the most sensitive probe (Fig. 1B) (data not shown for the others). To further improve the sensitivity of the detection of Ras activation in the plasma membrane, an intermolecular variant of the MP-targeted RasBRET probe (the energy donor and acceptor are in different polypeptide chains; MP-YFP-Ras + RBD-luciferase) (see “Experimental Procedures” and Fig. 1C) was constructed. Fig. 1C shows that this setup yielded a greatly improved and very sensitive probe for measuring Ras activation. The higher signal-to-noise ratio of the intermolecular biosensor can be explained by the high basal BRET signal of the intramolecular probes (∼1.4 versus ∼1.1) because, in these constructs, the donor and acceptor are in the same molecule. The cytosolic (spatially unbiased) intermolecular plasmid pair for RasBRET (non-targeted YFP-Ras plus RBD-luciferase) had the lowest absolute level of the BRET signal (∼0.9).

We validated that the change in the BRET signal using the intermolecular MP-RasBRET probe in our experimental protocol is dependent on the activation state of Ras (hence, the interaction of Ras and the Raf RBD) by performing measurements with mutated Ras domains in the probe. Fig. 2 shows that, by utilizing the “active” form of the intermolecular MP-RasBRET probe pair (G12V), we detected the maximal absolute level of the BRET signal. In contrast, when we applied the “inactive” Ras S17N mutation, the extent of the BRET signal was lower than the base line of the wild-type probe, showing that Ras was partially active in control cells. Because the optimization of BRET biosensors showed that the intermolecular MP-RasBRET probe resulted in the best Ras activation biosensors, we used the intermolecular BRET approach in our later work, and therefore, all subsequent data represent results using intermolecular BRET biosensors.

Ras Activation in the Plasma Membrane after AngII Stimulation of Different AT₁-R Mutants

After optimization and validation of our BRET strategy, experiments were performed with RasBRET probes in HEK293 cells cotransfected with the wild-type AT_1A-R, the tail deletion AT_1A-RΔ319 mutant (which is unable to internalize) (40), or the AT₁-R-DRY/AAY mutant (which is unable to couple to Gα_q and initiate inositol phosphate signaling) (35, 37). As shown in Fig. 3, AngII stimulation of the wild-type AT₁-R caused transient Ras activation in the plasma membrane, which was rapidly terminated because of the receptor desensitization and internalization. However, when the desensitization or internalization was reduced in cells expressing the AT_1A-RΔ319 mutant, Ras activation was enhanced and prolonged. We did not detect significant Ras activation when utilizing the AT₁-R-DRY/AAY mutant, which suggests that the AngII-induced Ras activation is predominantly G protein-mediated.

Subcellular Localization of Ras Activation

In the next set of experiments, the effect of extracellular stimuli on intracellular Ras activation was examined. To investigate specifically the possible compartmentalized Ras signaling processes, we targeted our probes to the surface of intracellular organelles using previously described targeting motifs (29). These motifs were placed at either the N or C terminus of the respective probes as described under “Experimental Procedures.” We used intermolecular probe pairs in which one of the interacting partners (either YFP-Ras or RBD-luciferase) was tagged with the targeting sequence while the other polypeptide was not targeted to organelles and was therefore expressed in the cytosol. The micrographs (confocal microscopic images) in Fig. 4 demonstrate that all probes were properly expressed in the targeted compartment. HEK293 cells were cotransfected with the AT_1A-R (wild-type AT_1A-R, AT_1A-RΔ319 or AT_1A-R-DRY/AAY) and with the indicated intermolecular probes targeted to various compartments, such as the trans-Golgi network (TGN), the ER, and endosomes.

EGF- and AngII-induced Ras activation was detected in the Golgi using YFP-Ras targeted to the TGN (the targeting sequence was fused to the N terminus of YFP-Ras, and RBD-luciferase was cytosolic) (Fig. 4A). AngII caused a rapid and transient Ras activation in the TGN (Fig. 4A). In contrast to the Ras activation in the plasma membrane, where AngII stimulation resulted in a much smaller response compared with EGF-evoked activation, the changes in the BRET ratios in the TGN were quite similar for EGF and AngII, suggesting that the relative Ras activation efficacy of AngII may be higher than that of EGF in this compartment. It is also important to note that the response to AngII consistently occurred more rapidly then the EGF-induced response. To assess the compartmentalized Ras signaling upon agonist stimulation, we compared the data obtained using the wild-type AT₁-R and an internalization-deficient AT₁-R mutant (AT_1A-RΔ319) coexpressed with various BRET probes in HEK293 cells. Our data show that the AngII-induced Ras activation in the TGN is not a consequence of receptor internalization because the internalization-deficient AT₁-R mutant showed prolonged, rather than inhibited, Ras activation compared with the wild-type AT₁-R (Fig. 4A).

We also detected Ras activation on the surface of the ER using both Ubc6- and Sac1- targeted intermolecular probes. In these constructs, the ER-targeting sequences were fused to the C terminus of the RBD-luciferase part while the YFP-Ras part was cytosolic. Interestingly, only AngII stimulation activated Ras in the ER, whereas EGF was not able to show this effect (Fig. 4B). The kinetics and characteristics of the AngII-induced Ras activation in the ER were indistinguishable when Sac1- or Ubc6-targeting sequences were used (Fig. 4B; only the results with the Sac1-targeted probe are shown). These data also suggest that the AngII-induced response is independent of EGF receptor transactivation. Similar to Ras activation detected in the TGN, the AngII-induced Ras activation was more sustained in the ER in cells expressing the internalization-deficient AT_1A-RΔ319 mutant compared with the wild-type receptor.

Although EGF receptor activation in endosomes was detected by resonance energy transfer measurements in previous studies (11), our RasBRET probe targeted with the FYVE domain to endosomes (the targeting sequence was fused to the C terminus of RBD-luciferase while YFP-Ras is cytosolic) was not able to detect EGF- or AngII-induced Ras activation in endosomes (Fig. 4C). Based on these data, it is possible that endosomal Ras activation occurs away from the targeted phosphatidylinositol 3-phosphate-containing microdomain of this organelle.

Mechanism of AngII-induced Ras Activation

In the cells of the cardiovascular system, AngII-induced Ras activation (and hence, MAPK activation) is mostly the consequence of the transactivation of receptor tyrosine kinases, such as the EGF receptor. However, in some other cell types, AngII-induced ERK activation is independent of EGF receptor transactivation (17). It has been demonstrated that phosphorylation of ERK1/2 upon AngII stimulation in HEK293 cells, but not in C9 hepatic cells, is EGF receptor transactivation-independent, but Ras activation was not investigated (20). In our experiments using HEK293 cells, the EGF receptor kinase inhibitor AG1478 abolished the EGF-evoked Ras activation in the plasma membrane, but it had no inhibitory effect on the AngII-induced Ras activation in the plasma membrane (Fig. 5A). The AngII-induced Ras activation in the TGN was not affected (Fig. 5 B). These data suggest that Ras activation in HEK293 cells is primarily independent of EGF receptor transactivation but requires G protein coupling because AngII stimulation of cells expressing the AT₁-R-DRY/AAY mutant failed to initiate Ras signaling in the plasma membrane (Fig. 3) and in the intracellular organelles studied (data not shown).