Arrestin-dependent Angiotensin AT1 Receptor Signaling Regulates Akt and mTor-mediated Protein Synthesis*

Background: Angiotensin AT1 receptors use G protein-independent signals to stimulate protein synthesis. Results: Arrestin-dependent activation of ERK1/2 and Akt signaling regulates mTOR-p70/85S6K and p90RSK, leading to increased protein translation in HEK293 and primary vascular smooth muscle. Conclusion: Arrestin-dependent ERK1/2 and Akt signaling cooperatively regulate cell growth. Significance: Arrestin pathway-selective AT1 receptor agonists stimulate Akt-mTOR signaling and protein translation. Control of protein synthesis is critical to both cell growth and proliferation. The mammalian target of rapamycin (mTOR) integrates upstream growth, proliferation, and survival signals, including those transmitted via ERK1/2 and Akt, to regulate the rate of protein translation. The angiotensin AT1 receptor has been shown to activate both ERK1/2 and Akt in arrestin-based signalsomes. Here, we examine the role of arrestin-dependent regulation of ERK1/2 and Akt in the stimulation of mTOR-dependent protein translation by the AT1 receptor using HEK293 and primary vascular smooth muscle cell models. Nascent protein synthesis stimulated by both the canonical AT1 receptor agonist angiotensin II (AngII), and the arrestin pathway-selective agonist [Sar1-Ile4-Ile8]AngII (SII), is blocked by shRNA silencing of βarrestin1/2 or pharmacological inhibition of Akt, ERK1/2, or mTORC1. In HEK293 cells, SII activates a discrete arrestin-bound pool of Akt and promotes Akt-dependent phosphorylation of mTOR and its downstream effector p70/p85 ribosomal S6 kinase (p70/85S6K). In parallel, SII-activated ERK1/2 helps promote mTOR and p70/85S6K phosphorylation, and is required for phosphorylation of the known ERK1/2 substrate p90 ribosomal S6 kinase (p90RSK). Thus, arrestins coordinate AT1 receptor regulation of ERK1/2 and Akt activity and stimulate protein translation via both Akt-mTOR-p70/85S6K and ERK1/2-p90RSK pathways. These results suggest that in vivo, arrestin pathway-selective AT1 receptor agonists may promote cell growth or hypertrophy through arrestin-mediated mechanisms despite their antagonism of G protein signaling.

ribosomes to regulate protein translation. All three steps of protein synthesis require a unique set of translation factors: the eukaryotic initiation, termination, and release factors; eIFs, eEFs, and eRFs, respectively. The Akt and ERK pathways ultimately converge on these translation factors; both p70S6K and p90RSK phosphorylate eIF4B at Ser-422 (6) and eEF2 kinase at Ser-366 (7).
GPCRs, like the angiotensin AT1 receptor, have been shown to regulate both ERK1/2 and Akt through non-canonical G protein-independent signaling mechanisms involving arrestins. Arrestin-dependent activation of ERK1/2 is one of the proto-typic arrestin-mediated signals (8). Ligand-dependent recruitment of arrestin to the receptor promotes assembly of a stable cRaf1-MEK-ERK1/2 signalsome complex, leading to activation of a discrete cytosolic pool of activated ERK1/2 (8,9). Signalsome-bound ERK1/2 cannot translocate to the nucleus and is transcriptionally silent (10,11), but has been reported to promote phosphorylation of cytosolic ERK1/2 substrates, including p90RSK and MNK-1, and to stimulate protein synthesis (12,13).
Given the capacity of arrestins to regulate both the ERK1/2 and Akt pathways via GPCR-arrestin signalsomes, we hypothesized that they may function in the control of protein translation by integrating the activity of the two principal pathways controlling mTORC1 (Fig. 1B). We find that both angiotensin II (AngII) and the arrestin pathway-selective AT 1 receptor agonist, [Sar 1 -Ile 4 -Ile 8 ]AngII (SII) (7,19), stimulate mTORC1-dependent protein translation in HEK293 and primary vascular smooth muscle cells. The responses are arrestin-dependent, and require both ERK1/2 and Akt activity. Both Akt and ERK1/2 contribute to activation of the mTORC1-p70S6K axis, while ERK1/2 is the principal activator of the MAPKAPK, p90RSK. These data underscore the pivotal role of arrestins in G protein-independent AT 1 receptor signaling and identify novel properties of arrestin pathway-selective agonist ligands.
Cell Culture and Transfection-HEK293 cells stably expressing the rat AT 1A receptor (21) were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum, and 1% antibiotic/antimycotic solution. Prior to experimentation, cells were serum-deprived overnight in serum-free growth medium supplemented with 0.1% bovine serum albumin and 1% antibiotic/antimycotic solution. Transient transfection of HEK293 cells for flag-␤arrestin2 immunoprecipitation was performed in 10 cm dishes (8 million cells/ dish) using FuGENE 6 according to the manufacturer's instructions with 10 g of plasmid DNA per dish and 3 l of FuGENE 6 per g of DNA.
Primary rat aortic VSMCs were isolated and maintained as described previously (22). VSMCs were cultured in minimum essential medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution. Cells were fed every 2 days and subcultured upon reaching 90% confluence. Prior to each experiment, cells were incubated for 24 to 48 h in serumfree growth medium supplemented with 0.1% bovine serum albumin and 1% antibiotic/antimycotic solution. All experiments employing primary VSMC were performed between passages four and nine.
Silencing ␤arrestin1/2 Expression by RNA Interference-The HEK293 FRT/TO ␤arrestin1/2 shRNA cell line carrying tetracycline-inducible shRNA simultaneously targeting the ␤arrestin1 and 2 isoforms (5Ј-CGTCCACGTCACCAACAAC-3Ј) was generated as previously described (20). Transcription of shRNA was induced by 48 h exposure to 1 M doxycycline and down-regulation of ␤arrestin1/2 was documented by immunoblotting. Alternatively, transient down-regulation of ␤arrestin1/2 was performed using 19-nucleotide duplex siRNA with 3Ј dTdT overhangs. The sequence 5Ј-ACCUGCGCCUUCCGCUAUGTT-3Ј was used for silencing and the non-silencing RNA duplex 5Ј-AAUUCUCCG-AACGUGUCACGU-3Ј was used as the negative control. HEK293 cells at 50% confluence in 10 cm plates were transfected with 6 g of control or ␤arrestin1/2-targeted siRNA using 50 l of Gene-Silencer TM according to the manufacturer's instructions. Assays were performed 72 h after gene silencing. Silencing of ␤arrestin1/2 expression was confirmed by immunoblotting.
For lentiviral down-regulation of ␤arrestin1/2 expression, VSMCs were grown to 70 -80% confluence then treated at a MOI of 50 with a 1:1 mixture of ␤arrestin1 and ␤arrestin2 shRNA lentiviral particles or cop GRP control lentiviral particles for 8 h in serum-free medium. Following infection, cells were incubated in complete medium for 24 h then incubated for 48 h in serum-free growth medium containing 0.1% BSA prior to treatment with 50 M SII for the times indicated. Silencing of ␤arrestin1/2 expression was confirmed by immunoblotting.
Protein Translation-The rate of net de novo protein synthesis was determined by L-[ 3 H]leucine pulse labeling. Subconfluent cells in 6-cm dishes were given a 2 h pulse of 5 Ci of L-[ were washed three times with ice-cold 0.5 N perchloric acid and resuspended in 0.3 N NaOH at 37°C for 1 h, after which 25 l of protein extract was added to 5 ml of scintillation mixture and 3 H dpm incorporated into protein determined by scintillation counting. Nonspecific counts measured in parallel plates that were lysed immediately following exposure to L-[ 3 H]leucine were background subtracted, and specific counts were normalized to total cellular protein determined by bicinchoninic acid protein assay (Pierce/Thermo Fisher Scientific).
Alternatively, protein translation rates were determined using the Click-iT AHA Alexa Fluor 488 Protein Synthesis High Content Screening Assay (Molecular Probes/Invitrogen, Grand Island, NY). Cells grown to subconfluence in 96-well plates were stimulated for 2 h as appropriate, and metabolically labeled for the last 30 min of incubation with the azide-containing amino acid, L-azidohomoalanine. Cells were then fixed and permeabilized, and labeled nascent protein was visualized using Click-iT chemistry that covalently attaches the fluorophore Alexa-488 to L-azidohomoalanine according to the manufacturer's protocol. Nuclei were counterstained using Hoechst 33342 dye. Cell monolayers were then imaged using an IN Cell High Content Analyzer (GE Healthcare Life Sciences, Pittsburgh, PA) to measure total levels of fluorescence in each field. Alexa-488 fluorescence was divided by the Hoechst 33342 fluorescence to normalize for cell number between fields.
Because of its high molecular weight, samples for mTOR immunoblotting were resolved on 4 -12% Bis-Tris Gels (Invitrogen) run using 3-(N-morpholino)-propanesulfonic acid buffer and transferred to Invitrolon PVDF membranes using the wet-tank method. Membranes were blocked overnight in 5% milk in Tris-buffered saline-Tween 20 buffer. Primary antibodies were diluted 1:1000 in Tris-buffered saline-Tween 20 buffer and incubated overnight at 4°C. Membranes were washed five times and incubated with horseradish peroxidaseconjugated secondary antibody in Tris-buffered saline-Tween 20 buffer for 1 h at room temperature prior to development.

RESULTS
AT 1A Receptor-stimulated Protein Translation Requires ␤arrestin1/2, ERK1/2, and Akt Activity-The native AT 1 receptor ligand, AngII, initiates the full spectrum of AT 1 signaling, engaging both heterotrimeric G protein-and arrestin-regulated effectors. In contrast, the substituted octapeptide, SII (19), is an arrestin pathway-selective AT 1 agonist with greatly diminished efficacy for heterotrimeric G protein activation, but near native ability to recruit arrestins and promote receptor internalization (9,18,19,23). Despite its lack of G protein efficacy, SII has been shown in global phosphoproteomic studies to elicit a robust protein phosphorylation response (18,24,25).
To determine the role of arrestins, ERK1/2, and Akt in AT 1 receptor-stimulated protein translation, we employed both AngII and SII, since the former reflects physiological AT 1 activation, while the latter pharmacologically isolates the G protein-independent aspects of AT 1 signaling. As shown in Fig. 2, the two ligands provoked similar increases in protein translation rate in HEK293-AT 1A cells measured either by L-[ 3 H]leucine ( Fig. 2A) or L-azidohomoalanine ( Fig. 2B) pulse labeling. AT 1 receptor-mediated stimulation of protein synthesis was blocked by the MEK1/2 inhibitor, PD98059, indicating that ERK1/2 is required for the response. Ligand-activated, but not basal, protein translation was also completely blocked by the PDK1 inhibitor, OSU03012. PDK1, which phosphorylates Akt on residue Thr-308, is the proximal kinase responsible for Akt activation.
To test whether the AngII or SII effects involved arrestins, we employed HEK293 FRT/TO ␤arrestin1/2 shRNA cells that were transiently transfected with the rat AT 1A receptor expression plasmid. Forty-eight h exposure of these cells to doxycycline results in 70 -90% down-regulation of ␤arrestin1/2 expression (Fig. 2B). As shown, stimulation of protein translation by both AngII and SII was blocked by ␤arrestin1/2 silencing, suggesting that the AT 1A receptor predominantly uses arrestin-mediated pathways to couple to the translational machinery.
AT 1A Receptor Activation of Arrestin-bound Akt Is Independent of ERK Activation-Since the protein translation response to AngII and SII was co-dependent upon ERK1/2 and Akt activity, we next tested whether these two arrestin-regulated kinase cascades interacted proximally. As we previously described (18), basal whole cell levels of Akt phosphorylation in HEK293 cells are constitutively high, necessitating that the phosphorylation state of the arrestin-bound Akt pool be measured in ␤arrestin2 immunoprecipitates. Fig. 3A shows the effects of PD98059 and OSU03012 on basal Akt phosphorylation in HEK293-AT 1A cells. As expected, no significant change in total cellular phospho-Akt was measurable in response to AngII or SII exposure (18). Importantly, while the PDK1 inhibitor, OSU03012, dramatically reduced global Akt phosphorylation, the MEK1/2 inhibitor PD98059 had no effect, demonstrating that it did not nonspecifically affect Akt activity at the concentration employed. To determine the effect of agonist stimulation on the arrestin-bound pool of Akt, HEK293-AT 1A cells were transiently transfected with plasmid encoding flag-␤arrestin2, and Akt phosphorylation was measured in flag-␤arrestin2 immunoprecipitates. As shown in Fig. 3B, both SII and AngII significantly increased the level of ␤arrestin2-bound phospho-Akt. The response was unaffected by ERK1/2 pathway inhibition, but was completely blocked by OSU03012, suggesting that arrestin-dependent activation of Akt is independent of concomitant ERK1/2 activation.
Arrestin-dependent ERK1/2 and Akt Signaling Converges on p70S6K-Since arrestin-dependent activation of ERK1/2 and Akt appear to be largely independent at the signalsome level, yet both are required to stimulate protein translation, we next determined their contribution to regulation of the p70/85S6K and p90RSK pathways leading to translation initiation (Fig. 1A).
Arrestin-dependent ERK1/2 and Akt Signaling Regulates mTORC1 Activation and Function-Although arrestin-dependent ERK1/2 signaling appeared to be sufficient to activate p90RSK, SII-mediated activation of p70/85S6K required both ERK1/2 and Akt. Since mTORC1 is the critical upstream regulator of p70S6K, and is itself regulated by both ERK1/2 and Akt (Fig. 1A), we next tested whether the two arrestin signaling pathways converged at the level of mTORC1.
consistent with the known modulatory role of ERK1/2-p90RSK. Since p70/85S6K is a direct mTOR substrate, we tested whether AT 1A receptor-mediated phosphorylation p70/85S6K was mTOR-dependent. As shown in Fig. 5B, SII-induced p70/ 85S6K phosphorylation was blocked by pretreatment with either rapamycin, an inhibitor of the raptor-containing mTORC1, or with PP242, a dual inhibitor of mTORC1 and the rictor-containing mTORC2, suggesting that the G protein-independent co-regulation of p70/85S6K by ERK1/2 and Akt occurs primarily at the level of mTOR. In contrast, AngII-mediated p70/85S6K phosphorylation was not significantly affected by rapamycin and while significantly reduced, was not abolished, by PP242, suggesting that the AngII response is less dependent on mTORC1 than the more arrestin-dependent SII response. This ability to bypass mTOR may reflect the enhanced efficacy of AngII for ERK1/2 activation (Fig. 3C), since ERK1/2 can reportedly phosphorylate p70S6K directly (27), and AngII-stimulated p70/85S6K phosphorylation is markedly inhibited by PD98059 in HEK293-AT 1A cells (Fig.  4A).
We next tested whether AT 1A receptor-stimulated protein translation required mTORC1. As shown in Fig. 5C, treatment of HEK293 AT 1A cells with either rapamycin or PP242 completely inhibited SII-and AngII-stimulated of L-[ 3 H]leucine incorporation. Identical results were obtained using the L-azidohomoalanine incorporation method (Fig. 5D).
As shown in Fig. 6C, AngII-and SII-mediated Akt phosphorylation was detectable within 1 min of stimulation, peaked at 5 min, and returned nearly to baseline by 30 min. Ang II-stimulated ERK1/2 phosphorylation followed a similar time course, while SII-stimulated ERK1/2 phosphorylation was slightly delayed in onset, consistent with prior reports (29). AngII stim-   Representative phospho-p70/ 85S6K immunoblots are shown above a bar graph depicting the Mean Ϯ S.E. of three biological replicates. Glyceraldehyde phosphate dehydrogenase (GAPDH) was immunoblotted as a protein loading control. B, effect of ␤arrestin1/2 down-regulation on whole cell p70/85S6K phosphorylation. HEK293-AT 1A cells were transiently transfected with plasmid DNA encoding siRNA targeting ␤arrestin1/2 as described. Following serum-deprivation, cells were stimulated with AngII or SII for 5 min. Representative phospho-p70/ 85S6K and ␤arrestin1/2 immunoblots are shown above a bar graph depicting the mean Ϯ S.E. of three biological replicates. GAPDH was immunoblotted as a protein loading control. C, effect of ERK1/2 and Akt inhibitors on whole cell p90RSK phosphorylation. Serum-deprived HEK293-AT 1A cells were pretreated with PD98059 or OSU03012 prior to 5 min stimulation with AngII or SII. Representative phospho-p90RSK immunoblots are shown above a bar graph depicting the mean Ϯ S.E. of three biological replicates. GAPDH was immunoblotted as a protein loading control. In all panels; * greater than nonstimulated (NS), p Ͻ 0.05, n ϭ 3; † less than in the absence of inhibitor, p Ͻ 0.05, n ϭ 3. ulated ERK1/2 and Akt phosphorylation with similar potency (EC 50 ϳ0.4 nM; Fig. 6D). Consistent with its known 200-fold lower affinity for the AT 1A receptor (19,30), SII was much less potent than AngII. In addition, the EC 50 of SII for ERK1/2 activation was about 5-fold higher than for Akt activation (ERK1/2 EC 50 ϭ 470 nM versus Akt EC 50 ϭ 85 nM), suggesting differences in the efficiency of coupling to the two pathways.
We next tested whether the SII-stimulated Akt and ERK1/2 responses were sensitive to PD98059 and LY294002 in VSMCs. As in HEK293-AT 1A cells, SII-stimulated Akt phosphorylation was unaffected by the MEK1 inhibitor, but abolished by PI3K inhibition (Fig. 6E). Conversely, SII-stimulated ERK1/2 activation was sensitive to PD98059, but not LY294002. The attenuation of SII-stimulated ERK1/2 activation by LY294002 seen in HEK293-AT 1A cells (Fig. 3D) did not occur in VSMC, underscoring that arrestin-dependent regulation of ERK1/2 and Akt signaling are largely independent. AngII-stimulated Akt and ERK1/2 phosphorylation exhibited a similar pattern of inhibitor sensitivity.
As shown in Fig. 7A, 2 h stimulation of VSMCs with SII or AngII produced a significant increase in nascent protein abundance assayed by L-azidohomo-alanine incorporation. The response was blocked by cycloheximide, demonstrating that it was due to increased de novo protein translation. To test whether the response was arrestin-dependent in a native cell background, we employed lentiviral vectors to deliver shRNA targeting ␤arrestin1/2 (Fig. 7B). Down-regulating ␤arrestin1/2 expression by 60 -70% led to an increase in basal protein translation compared with VSMC treated with control lentivirus. As in HEK293 FRT/TO ␤arrestin1/2 shRNA cells, neither AngII nor SII stimulated protein translation following ␤arrestin1/2 down-regulation. In contrast, epidermal growth factor, which acts via a classical receptor tyrosine kinase, provoked similar net increases in protein translation in VSMC treated with either control or ␤arrestin1/2 shRNA lentivirus. Since ␤arrestin expression has been shown to tonically inhibit Akt-GSK3␤ signaling in dopaminergic neurons (14 -16), the increase in basal protein translation seen upon ␤arrestin1/2 down-regulation  may reflect de-repression of basal Akt activity. At the same time, loss of ␤arrestin scaffolds appears to uncouple AT 1 , but not epidermal growth factor, receptors from the translational machinery.
To determine the contribution of G protein-independent signaling to endogenous AT 1 receptor-stimulated protein translation, we tested whether the SII response was sensitive to ERK1/2, Akt, and mTORC1 inhibition. Consistent with our findings in HEK293-AT 1A cells, exposing rat VSMCs to PD98059, the phosphatidylinositol 3-kinase inhibitor LY294002, rapamycin, or PP242 completely blocked SIIstimulated L-azidohomo-alanine incorporation (Fig. 7C). The native agonist, AngII, also demonstrated co-dependence on ERK1/2, Akt, and mTORC1.

DISCUSSION
Regulation of the protein translational machinery by extracellular signals involves complex interplay between the ERK1/2-p90RSK and Akt-mTORC1-p70S6K pathways that regulate ribosomal eIFs, eEFs, and eRFs (Fig. 1A). Our data demonstrate that arrestin-dependent signals regulating both ERK1/2 and Akt downstream of the angiotensin AT 1A receptor are critical mediators of GPCR-regulated protein translation.
The co-regulation of protein translation by arrestin-dependent signaling is best demonstrated using SII, since this arrestin pathway-selective AT 1A receptor agonist is devoid of significant G protein efficacy (18,19,23). SII elicits a protein translation response that is comparable in magnitude to that produced by AngII, and completely blocked by shRNA-mediated downregulation of ␤arrestin1/2. As previously reported (12,13), activation of p90RSK by SII is ERK1/2 dependent, in that it is abolished by MEK1/2-ERK1/2 inhibition and not significantly affected by PDK1-Akt inhibition. In contrast, both ERK1/2 and Akt signaling are involved in activation of the mTORC1-p70/ 85S6K pathway. The two signals converge at the level of mTORC1, since inhibiting either is sufficient to impair mTORC1 Ser-2448 phosphorylation, p70/85S6K Thr-421/ FIGURE 6. SII independently stimulates ERK1/2 and Akt activation in primary rat aortic VSMCs. A, effect of AngII or SII on ERK1/2 and Akt phosphorylation. Serum-deprived VSMCs were stimulated for 5 min with AngII (100 nM) or SII (50 M) prior to performing phospho-Akt and phospho-ERK1/2 immunoblots on whole cell lysates. In each panel, representative immunoblots are shown above a bar graph depicting the Mean Ϯ S.E. of three biological replicates. B, down-regulating ␤arrestin1/2 expression attenuates SII-mediated Akt phosphorylation. Rat aortic VSMCs were infected with lentiviral vectors encoding shRNA targeting ␤arrestin1/2 or control lentivirus. After serum deprivation, cells were stimulated for 5 min with SII prior to performing phospho-Akt immunoblots on whole cell lysates. A representative phospho-Akt mmunoblot is shown above a bar graph depicting the Mean Ϯ S.E. of three biological replicates. GAPDH was immunoblotted as a protein loading control. A representative ␤arrestin immunoblot of control and ␤arrestin1/2 shRNA lentivirus treated VSMCs is shown to the right. C, time course of AngII-and SII-stimulated Akt and ERK1/2 phosphorylation. Serum-deprived rat aortic VSMCs were stimulated for the indicated times with AngII (100 nM) or SII (50 M) prior to performing phospho-Akt and phospho-ERK1/2 immunoblots on whole cell lysates. In each panel, representative immunoblots are shown above a bar graph depicting the mean Ϯ S.E. of three biological replicates. D, dose dependence of AngII-and SII-stimulated Akt and ERK1/2 phosphorylation. Serum-deprived rat aortic VSMCs were stimulated for 5 min with the indicated concentrations of AngII or SII prior to performing phospho-Akt and phospho-ERK1/2 immunoblots on whole cell lysates. In each panel, representative immunoblots are shown above a bar graph depicting the mean Ϯ S.E. of three biological replicates. EC 50 values were derived by curve fitting using GraphPad Prism software. E, effect of MEK and PI3K inhibitors on AngII and SII-stimulated Akt and ERK1/2 phosphorylation. Serum-deprived rat aortic VSMCs were pretreated with PD98059 (20 M; 1 h) or LY294002 (10 M; 1 h) prior to 5 min stimulation with AngII or SII. Levels of whole cell phospho-Akt and -ERK1/2 were determined by immunoblotting whole cell lysates. Representative immunoblots are shown above a bar graph depicting the mean Ϯ S.E. of three biological replicates. In all panels; * greater than non-stimulated (NS), p Ͻ 0.05, n ϭ 3; † less than in the absence of inhibitor, p Ͻ 0.05, n ϭ 3.
As with SII, stimulation of protein translation by AngII is both arrestin-and mTORC1-dependent. In HEK293-AT 1A cells, AngII and SII produce comparable activation of arrestinbound Akt, while AngII, which can activate both hetero-trimeric G protein and arrestin signaling, produces more robust ERK1/2 activation. While this enhanced ERK1/2 activation may render AngII-stimulated p70/85S6K phosphorylation less sensitive to Akt and mTORC1 inhibition, AngII-induced protein translation in both HEK293-AT 1A cells and primary VSMCs requires Akt and mTORC1 activity, indicating that the Akt-mTORC1-p70/85S6K pathway is as important for the native ligand as for SII.
The non-visual arrestins, ␤arrestin1/2, perform dual roles in modulating GPCR signaling; serving both as the principal mediators of GPCR desensitization and internalization, and as signal transducers that confer additional G protein-indepen-dent signaling capability (31). Many arrestin-dependent signals result from scaffolding protein kinase and/or phosphatase cascades within receptor-arrestin signalsomes (32). Of these, scaffolding of cRaf1-MEK-ERK1/2 and PP2A-Akt-GSK3␤ complexes is among the best characterized. Arrestins recruit a cRaf1-MEK-ERK1/2 complex to agonist-occupied GPCRs, leading to sustained activation of a spatially, temporally and functionally discrete ERK1/2 pool (8 -11, 33). Our present data support the hypothesis that one important function of this ERK1/2 pool is to regulate protein translation by activating the MAPKAPKs, p90RSK, and MNK-1, and serving as a co-activator of the mTORC1-p70/85S6K pathway.
AT 1A receptor-mediated activation of arrestin-bound Akt resulting from SII-or AngII-stimulated phosphorylation of I2PP2A, inhibition of PP2A, and relief of tonic PP2A-mediated inhibition of Akt in the signalsome complex (18). Our present data suggest that an important function of this arrestin-bound Akt pool is to activate the mTORC1-p70/85S6K pathway, which in conjunction with ERK1/2, leads to increased mTORC1-dependent protein translation.
Arrestin-dependent signaling has been shown to exert pleiotropic effects in cardiovascular tissues (36). Arrestin pathwayselective AT 1 receptor agonists have been reported to improve contractile parameters in isolated murine cardiomyocytes (37), to reduce blood pressure and improve cardiac performance in vivo (38), and to promote cardiomyocyte survival during acute cardiac injury (39). Indeed, an arrestin-biased angiotensin analog, TRV120023 (Sar-Arg-Val-Tyr-Lys-His-Pro-Ala-OH), that possesses higher affinity for the AT 1 receptor than SII, has been advanced as a potential therapeutic in cardiogenic shock (38 -40). On the other hand, ␤arrestin2-null mice exhibit reduced neointimal VSMC hyperplasia following endothelial injury, and SII stimulates VSMC proliferation, migration, and anti-apoptotic signaling in vitro (17,41,42), suggesting that arrestin-dependent AT 1 receptor signaling may promote atherosclerosis. Moreover, in the adrenal, ␤arrestin1 signaling underlies AngIIinduced aldosterone biosynthesis, and inhibition of adrenal ␤arrestin1 signaling in vivo attenuates post-myocardial infarction heart failure and adverse remodeling by inhibiting aldosterone-dependent salt retention (43,44). Our present data demonstrating a central role for arrestin-dependent ERK1/2 and Akt signaling in AT 1 receptor regulation of the mTOCR1 complex further suggests that arrestin-dependent signaling may promote adverse effects in the cardiovascular system by stimulating cellular growth, hyperplasia, and proliferation.