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β-Arrestins turn off G protein-mediated signals and initiate distinct G protein-independent signaling pathways. We previously demonstrated that angiotensin AT1 receptor-bound β-arrestin 1 is cleaved after Phe388 upon angiotensin II stimulation. The mechanism and signaling pathway of angiotensin II-induced β-arrestin cleavage remain largely unknown. Here, we show that protein Tyr phosphatase activity is involved in the regulation of β-arrestin 1 cleavage. Tagging of green fluorescent protein (GFP) either to the N-terminus or C-terminus of β-arrestin 1 induced conformational changes and the cleavage of β-arrestin 1 without angiotensin AT1 receptor activation. Orthovanadate and molybdate, inhibitors of protein Tyr phosphatase, attenuated the cleavage of C-terminal GFP-tagged β-arrestin 1 in vitro. The inhibitory effects of okadaic acid and pyrophosphate, which are inhibitors of protein Ser/Thr phosphatase, were less than those of protein Tyr phosphatase inhibitors. Cell-permeable pervanadate inhibited angiotensin II-induced cleavage of β-arrestin 1 in COS-1 cells. Our findings suggest that Tyr phosphorylation signaling is involved in the regulation of angiotensin II-induced β-arrestin cleavage.
The angiotensin AT1 receptor belongs to the G protein-coupled receptor family and transduces diverse signals in G protein- and β-arrestin-dependent pathways (Oro et al., 2007). Upon angiotensin II binding to the angiotensin AT1 receptor, a conformational change in the receptor modulates G protein, Gq, and activates phospholipase Cβ for IP3 and Ca2+-mediated vasoconstriction (Oro et al., 2007). β-Arrestin 1 and 2, ubiquitously expressed in mammalian tissues, are recruited from the cytoplasm to the activated angiotensin AT1 receptor for clathrin-mediated endocytosis (Krupnick et al., 1997). In addition to the role in desensitization and endocytosis, β-arrestins form scaffolds for a number of signaling molecules in MAPK (mitogen-activated protein kinase) cascades and non-receptor Tyr kinases for G protein-independent signals (Lefkowitz and Shenoy, 2005). The β-arrestin 1 and 2 are comprised of N- and C-domains of equal size (Han et al., 2001), with the N-domain serving as a phosphate sensor recognizing the phosphorylated G protein-coupled receptor (Nobles et al., 2007) and the C-domain acting as adaptor binding sites for clathrin and AP-2 (Lin et al., 1997). Previous studies have shown that there are distinct inactive and active conformations in β-arrestin. In the inactive state, the polar core between the N- and C-domain of β-arrestin 1 is kept intact with the C-terminus buried in the structure (Han et al., 2001). However, once β-arrestin 1 is bound to the phosphorylated receptor, a conformational change in β-arrestin occurs and extrudes its C-terminal region towards the outside, thus enabling the binding of clathrin and AP-2 for clathrin-mediated endocytosis (Lefkowitz and Shenoy, 2005). β-Arrestin 1 and 2 have distinct active conformations when bound to the activated receptor (Nobles et al., 2007; Xiao et al., 2004).
In a previous study, we showed that a fraction of β-arrestin 1 bound to the angiotensin AT1 receptor is cleaved upon receptor activation and it requires stable interaction between the angiotensin AT1 receptor and β-arrestin (Lee et al., 2008). Angiotensin II and inverse agonist EXP3174 induced cleavage at distinct sites on β-arrestin 1; after Phe388 upon angiotensin II treatment and after Pro276 upon EXP3174 treatment, respectively. These results suggested ligand-induced selectivity in β-arrestin-mediated signaling. With distinct cleavage sites of β-arrestin 1 and 2 by angiotensin II, our data also demonstrated that the receptor-bound active conformations of β-arrestin 1 and 2 are different. The biological significance and the signaling pathway of ligand-induced β-arrestin cleavage, however, are largely unknown.
In this study, we sought to elucidate the regulatory mechanism of angiotensin II-mediated β-arrestin 1 cleavage in COS-1 cells expressing the angiotensin AT1 receptor. Since protein kinase activation has been implicated in the activation of proteases downstream of angiotensin AT1 receptor (Eguchi et al., 2001), we hypothesized that β-arrestin proteolysis is regulated by cellular protein kinases or phosphatases. We used inhibitors of protein Tyr or Ser/Thr phosphatase to investigate the effect on β-arrestin proteolysis. Here, we show that cell-permeable pervanadate inhibits angiotensin II-induced cleavage of β-arrestin 1 in COS-1 cells. Our finding suggests that protein Tyr phosphatase activity is involved in the regulation of G protein-coupled receptor-engaged β-arrestin proteolysis.
Angiotensin II was purchased from Bachem (USA). Lipofectamine 2000 was purchased from Invitrogen (USA). Monoclonal antibodies to myc and β-arrestin 2 (H-9) were purchased from Santa Cruz Biotechnology, Inc (USA). Since the anti-β-arrestin 2 antibody (H-9) recognized both β-arrestin 1 and 2 in our previous studies (Lee et al., 2007; 2008), we used this antibody for the detection of transfected β-arrestin 1 in this study. Monoclonal antibody to GFP was purchased from Clontech (USA). Horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody was purchased from Upstate (USA). COS-1 cells were purchased from American Type Culture Collection (USA). Western blot stripping buffer was purchased from Pierce (USA). All other reagents, unless stated otherwise, were from Sigma (USA).
Three fusion constructs with myc or GFP-tagged β-arrestin 1 were generated by polymerase chain reaction by the following primers using pfu DNA polymerase. For Myc-β-arrestin 1, the forward primer was 5′-AACCGGATCCGATGGGCGACAAAGGGAC-3′ (BamHI site underlined, and the N-terminus of β-arrestin 1 in boldface type), and the reverse primer was 5′-AACCCTCGAGCTATCTGTCGTTGAGCCGCGGAG-3′ (XhoI site underlined, the C-terminus of β-arrestin 1 in boldface type). For the N-terminal GFP-tagged GFP-β-Arrestin 1, the cDNA for cloning was subcloned into pEGFP-C1 vector (Clontech, USA). The forward primer was 5′-AACCCTCGAGCCATGGGCGACAAAGGGAC-3′ (XhoI site underlined, and the N-terminus of β-arrestin 1 in boldface type), and the reverse primer was 5′-AACCGGATCCCTATCTGTCGTTGAGCCGCG-3′ (BamHI site underlined, and the C-terminus of β-arrestin 1 in boldface type). The C-terminal GFP tagged β-Arrestin 1-GFP was generated using pEGFP-N3 vector. The forward primer was 5′-CCCCCTCGAGTCTACCATGGGCGACAAAGGGAC-3′ (XhoI site underlined, and the N-terminus of β-arrestin 1 in boldface type), and the reverse primer was 5′-AACCGGATCCTCTGTCGTTGAGCCCGCGGAGAGC-3′ (BamHI site underlined, and the C-terminus of β-arrestin 1 in boldface type). Accuracy of the fusion constructs in the expression vector was confirmed by DNA sequence analysis.
The synthetic rat angiotensin AT1 receptor gene, cloned in the shuttle expression vector pMT3, was used for expression. To express the angiotensin AT1 receptor and β-arrestin 1, 60-65% confluent COS-1 cells were grown in 6-well plates and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. The cells were transfected with 2 μg of purified angiotensin AT1 receptor and β-arrestin 1 cDNA using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions.
The following protocol was used for the detection of cleaved β-arrestin fragment. Transfected cells, cultured for 48 h, were treated with 1 μM angiotensin II or mock. Cells were washed with cold PBS and centrifuged at 13,000 rpm for 1 min at 4°C. Cell pellets were lysed in 100 μl of lysis buffer (25 mM HEPES, pH 7.2, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100, 1 mM PMSF, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 10% glycerol) for 60 min. The Sigma protease inhibitor mixture (P2714) was added to the lysis buffer. Cell lysates were centrifuged at 13,000 rpm for 10 min at 4°C to remove cell debris. Protein concentration was measured using Bradford assay kit (Bio-Rad). 20 μg of total protein was dissolved in Laemmli's sample buffer, boiled for 5 min at 95°C, and separated by SDS-PAGE with a 10% separation gel. Following electrophoresis, the proteins were transferred to nitrocellulose membranes and then blocked for 1 h at room temperature in 5% non-fat dry milk and 0.1% Tween-20 in PBS, pH 7.4. Incubation with primary antibody was carried out overnight at 4°C. Following washes with PBS, incubation with HRP-labeled secondary antibody was carried out for 1 h at room temperature. The detection was made with enhanced chemiluminescence (GE Healthcare) and the films were scanned for densitometry analysis using Fuji Multiguage V3.0 Software (Fuji Film). For the data shown in Fig. 3, the experimental procedures described above were followed except that the lysis buffer was prepared without any phosphatase inhibitors. Each phosphatase inhibitor component was added separately into lysis buffer to a final concentration of 25 mM β-glycerophosphate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM okadaic acid, 1 mM sodium orthovanadate, or 1 mM sodium molybdate. Data analysis was performed using GraphPad Prism 4 (GraphPad Software). Student's t-test was used for statistical analysis in Figs. 1 and and33.
Pervanadate was prepared using a 2 min incubation of 100 mM sodium orthovanadate and hydrogen peroxide in distilled water, followed by dilution to the appropriate concentration in serum-free DMEM. The solutions were used within 1 h of preparation.
In our previous pull-down assays, we demonstrated that β-arrestin 1 bound to the angiotensin AT1 receptor is cleaved upon angiotensin II stimulation in rat aortic smooth muscle (A7r5) cells and COS-1 cells (Lee et al., 2008). To detect the cleavage of β-arrestin 1 without immunoprecipitation, samples were blotted with anti-β-arrestin antibody (H-9) after cell lysis, SDS-PAGE, and transfer to nitrocellulose membrane.
As shown in Fig. 1A, the cleaved β-arrestin 1 was detected and band intensity for the cleaved fragment was increased upon angiotensin II treatment (1.34 ± 0.13 fold, P < 0.05). To confirm the cleavage event, the N-terminal myc-tagged β-arrestin 1 was constructed and assayed for the cleavage with anti-myc or anti-β-arrestin antibody. Surprisingly, substantial amount of cleaved myc-β-arrestin 1 fusion protein was found in the absence of angiotensin AT1 receptor in transiently transfected COS-1 cells. We speculate that the myc fusion to the N-terminus of β-arrestin 1 induces a conformational instability in β-arrestin. The cleavage of myc-β-arrestin 1 was enhanced following angiotensin II treatment (1.26 ± 0.11 fold, P < 0.05, blotted with anti-β-arrestin antibody). In both Figs. 1A and 1B, fold-increase of the cleaved β-arrestin 1 fragment upon angiotensin II treatment were alike, suggesting involvement of a similar mechanism in the regulation of cleavage. Since the myc-tag is localized in the N-terminus of β-arrestin 1, the cleavage site is considered to be localized in the C-terminal region of β-arrestin 1 as was reported (Lee et al., 2008).
To investigate the effect of a fusion protein on conformational changes in β-arrestin 1 that could lead to subsequent cleavages, GFP fusion at the N-terminus or the C-terminus of β-arrestin 1 (82 kDa) was constructed, respectively. The expression of each GFP-tagged β-arrestin 1 construct alone in COS-1 cells induced cleavages in β-arrestin at distinct sites. The N-terminal GFP fusion protein was cleaved at three sites in β-arrestin 1 (see Fig. 2A). We assume that the cleavage at Phe388 in β-arrestin 1 is responsible for generating the ~78 kDa band, and the generation of the smaller band is due to cleavage at sites not defined yet in the N-terminal region of β-arrestin 1. The C-terminal GFP fusion showed a different pattern of cleavage (see Fig. 2B). However, it also included the anticipated cleavage in β-arrestin 1, Phe388 (~31 kDa band indicated by an arrow). When each of the two GFP-tagged β-arrestin 1 constructs were separately co-expressed with the angiotensin AT1 receptor in COS-1 cells and stimulated by angiotensin II, there was no increase of cleaved β-arrestin bands for either N-terminal or C-terminal GFP-tagged β-arrestin 1 (Figs. 2A and 2B). This suggests that GFP fusion induces an intrinsic conformational change in β-arrestin, thus destabilizing its polar core and releasing the buried C-terminus of β-arrestin towards the outside. This finding of GFP-induced conformational change in β-arrestin as with myc-tag is consistent with previous findings that ligand induces a conformational change in ligand-receptor-arrestin ternary complex leading to the cleavage of visual arrestin (Azarian et al., 1995) and β-arrestin (Lee et al., 2008).
We investigated whether inhibitors of protein Tyr or Ser/Thr phosphatase could inhibit the proteolysis of β-arrestin 1. We utilized the characteristic that, after cleavage, the C-terminal GFP-tagged β-arrestin 1 fusion protein yields more distinct band separation on Western blot than wild-type β-arrestin 1, resulting in the generation of a ~31 kDa fragment shown in Fig. 2B.
We examined the effect of Tyr or Ser/Thr phosphatase inhibitors on this cleavage. After preparing lysis buffer without any phosphatase inhibitors, we added β-glycerophosphate, sodium fluoride, sodium pyrophosphate, okadaic acid, sodium orthovanadate, or sodium molybdate into separate lysis buffers. To our surprise, sodium orthovanadate and sodium molybdate, both inhibitors of protein Tyr phosphatase, inhibited the cleavage of β-arrestin 1-GFP 43% (0.57 ± 0.10, P < 0.05) and 37% (0.63 ± 0.10, P < 0.01), respectively, as shown in Fig. 3, compared to when all phosphatase inhibitors were absent (fourth lane from left). Sodium pyrophosphate and okadaic acid, inhibitors of protein Ser/Thr phosphatase, inhibited the cleavage 35% (0.65 ± 0.05, P < 0.01) and 34% (0.76 ± 0.11, P < 0.05), respectively. However, sodium fluoride and β-glycerophosphate, which belong to Ser/Thr phosphatase inhibitors, had a lesser effect on the inhibition of β-arrestin 1 proteolysis, 9% (0.91 ± 0.10) and 21% (0.79 ± 0.19, P < 0.05), respectively. Sodium fluoride is also used as an inhibitor of acid phosphatase. When all phosphatase inhibitors were included in lysis buffer, the inhibitory effect was similar to the effect of sodium orthovanadate. Thus, our data suggests that protein Tyr phosphatase inhibitors, orthovanadate and molybdate, have larger inhibitory effects than protein Ser/Thr phosphatase inhibitors on the proteolysis of β-arrestin 1-GFP fusion protein.
Although we have observed in vitro that inhibitors of both protein Tyr phosphatase and protein Ser/Thr phosphatase are effective in the inhibition of β-arrestin 1-GFP fusion protein cleavages, its cellular effect on angiotensin II-induced β-arrestin 1 cleavage is unknown. Hence, we preincubated COS-1 cells expressing the angiotensin AT1 receptor and β-arrestin 1 with either cell-permeable pervanadate or okadaic acid for 30 min before angiotensin II treatment. Cells were stimulated with 1 μM angiotensin II for 1 h and harvested. During cell lysis phosphatase inhibitors described in “Materials and Methods” were included in the lysis buffer. Compared to angiotensin II-induced β-arrestin 1 cleavage shown in Fig. 1, the pretreatment of 200 μM and 500 μM pervanadate prevented the cleavage of angiotensin II-induced β-arrestin 1 in COS-1 cells (see Fig. 4A). Pervanadate itself did not interfere with the expression of β-arrestin 1 as shown in Fig. 4B. Contrary to the effect of pervanadate, pretreatment of COS-1 cells with cell-permeable okadaic acid did not prevent the proteolysis of β-arrestin 1. This data supports our hypothesis that protein kinase activation, especially through Tyr phosphorylation, is implicated in the activation of proteases downstream of angiotensin AT1 receptor signaling pathway leading to the cleavage of β-arrestin.
Next, we examined concentration-dependent inhibition of β-arrestin 1-GFP cleavage by protein Tyr phosphatase inhibitors using the ~31 kDa β-arrestin 1-GFP fragment. The attenuation of β-arrestin 1 cleavage correlated with increasing concentrations of orthovanadate and molybdate, 0.01 to 1 mM, as shown in Fig. 5. It was shown that protein tyrosine phosphatase activities in cytosolic fraction of cultured rat hepatocytes were inhibited with the IC50 values of 0.03-0.05 mM (Pugazhenthi et al., 1996). Our data indicates that the cleavage of β-arrestin 1 is inhibited by protein tyrosine phosphatase inhibitors in a dose-dependent manner and the IC50 values are between 0.1 to 0.5 mM for orthovanadate (see Fig. 5A) and close to 0.1 mM for molybdate (see Fig. 5B). Since we measured the band intensity of cleaved β-arrestin 1-GFP fragment rather than the enzymatic activities of protein tyrosine phosphatase, we speculate that the IC50 values of protein tyrosine phosphatase will be lower than what we measured in this study.
The experiments presented in this study show that protein Tyr phosphatase activity is involved in the regulation of β-arrestin cleavage. Although the biological significance of angiotensin II-induced β-arrestin cleavage is unclear, it raises the possibility that once β-arrestin is cleaved, β-arrestin-mediated internalization of angiotensin AT1 receptor may be attenuated. The angiotensin AT1 receptor mediates diverse hormonal signals, leading to vasoconstriction and water-electrolyte balance through G protein-dependent and β-arrestin-dependent signaling pathways (Oro et al., 2007).
The ligand-receptor induced conformational changes in β-arrestin disrupt the intramolecular interaction of the basic N-domain and acidic C-domain, which allows binding of a hydro-phobic portion of arrestin distal to Arg175 and subsequent release of the β-arrestin C-terminus (Vishnivetskiy et al., 1999). Our previous report shows that a specific conformation of ligand-receptor-β-arrestin ternary complex is required for the site-specific cleavage of β-arrestin 1 and the cleavage is independent of IP3 and Ca2+-mediated signaling pathway (Lee et al., 2008). It was shown that the N-domain of β-arrestin is protected from in vitro tryptic digestion upon binding to phosphorylated receptor C-terminal region (Nobles et al., 2007), but N-terminal GFP fusion of β-arrestin 1 in our study destabilized the structure of β-arrestin, thus allowing cleavages at additional sites in the N-terminal region of β-arrestin 1 (see Fig. 2A). The N-terminal myc-tag generated a cleavage in the C-terminal region of β-arrestin 1, which perhaps is caused by less steric hindrance in the N-domain by the small sized myc-tag. Although GFP fusion to β-arrestin has been shown not to interfere with the normal functioning of β-arrestin such as translocation and endocytosis (Barak et al., 1997), our data indicates that GFP fusion to the N-terminus or the C-terminus of β-arrestin 1 induces distinct conformational changes in β-arrestin 1 and subsequent proteolysis by protease(s). The appearance of cleaved β-arrestin bands in the absence of angiotensin AT1 receptor activation suggests that conformational changes in β-arrestin by GFP fusion occur intrinsically. Our data suggests that using C-terminal GFP-tagged β-arrestin in imaging studies might show the movement of GFP tethered with full-length β-arrestin as well as the cleaved short fragment (4 kDa). The two additional cleavage sites in the N-terminal region of β-arrestin shown in GFP-β-arrestin fusion protein need to be determined to elucidate whether the same protease or another protease with different specificity is involved in the cleavage of β-arrestin.
Phosphorylation plays a key role in determining whether cell signaling will increase or decrease the activity of a protein. Several kinases and phosphatases have been shown to interact with the angiotensin AT1 receptor including Janus kinase, c-Src, SHP-1 and SHP-2 (Marrero et al., 1996; 1998). Since adding phosphatase inhibitors in cell culture medium or lysis buffer alter the general phosphorylation status of a cell or cell lysates, our work is limited in elucidating the specific kinase or phosphatase involved in the cleavage of β-arrestin. However, our findings show that Tyr-mediated angiotensin AT1 receptor signaling, independent of IP3 and Ca2+-mediated signaling pathway, is involved in this process, and that inhibitors of protein Tyr phosphatase such as orthovanadate and molybdate need to be included in lysis buffer to prevent the cleavage of β-arrestin during lysis and further experimental steps. The attenuation mechanism of β-arrestin 1-GFP cleavage by inhibitors of protein Ser/Thr phosphatases in vitro is uncertain, but it was reported that there are protein Tyr phosphatases regulated by Ser phosphorylation (Brautigan and Pinault, 1993; Garton and Tonks, 1994; Strack et al., 2002).
In conclusion, we observed that protein Tyr phosphatase activity is involved in the regulation of angiotensin II-induced β-arrestin cleavage. Our findings suggest that Tyr-mediated angiotensin AT1 receptor signaling, independent of IP3 and Ca2+-mediated signaling pathway, leads to the proteolysis of receptor-bound β-arrestins. Future studies need to be focused on elucidating the biological significance of ligand-induced β-arrestin cleavage and the kinases, phosphatases, and proteases responsible for the cleavage.
This work was supported by a research grant (2005) from Daegu University (to S.-H. J.) and the National Institutes of Health Grant RO1 HL57470 (to S.S.K.). M.K. is the recipient of a Korea Science and Engineering Foundation S&T Graduate Scholarship. We thank Dr. Chang-Soo Hong for valuable discussions.