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• W1993265529 abstract "Although tyrosine kinases are critically involved in the angiotensin II (Ang II) type 1 (AT1) receptor signaling, how AT1 receptors activate tyrosine kinases is not fully understood. We examined the structural requirements of the AT1 receptor for transactivation of the epidermal growth factor (EGF) receptor (EGFR). Studies using carboxyl terminal-truncated AT1 receptors indicated that the amino acid sequence between 312 and 337 is required for activation of EGFR. The role of the conserved YIPP motif in this sequence in transactivation of EGFR was investigated by mutating tyrosine 319. Ang II failed to activate EGFR in cells expressing AT1-Y319F, whereas EGFR was activated even without Ang II in cells expressing AT1-Y319E, which mimics the AT1 receptor phosphorylated at Tyr-319. Immunoblot analyses using anti-phospho Tyr-319-specific antibody showed that Ang II increased phosphorylation of Tyr-319. EGFR interacted with the AT1 receptor but not with AT1-Y319F in response to Ang II stimulation, whereas the EGFR-AT1 receptor interaction was inhibited in the presence of dominant negative SHP-2. The requirement of Tyr-319 seems specific for EGFR because Ang II-induced activation of other tyrosine kinases, including Src and JAK2, was preserved in cells expressing AT1-Y319F. Extracellular signal-regulated kinase activation was also maintained in AT1-Y319F through activation of Src. Overexpression of wild type AT1 receptor in cardiac fibroblasts enhanced Ang II-induced proliferation. By contrast, overexpression of AT1-Y319F failed to enhance cell proliferation. In summary, Tyr-319 of the AT1 receptor is phosphorylated in response to Ang II and plays a key role in mediating Ang II-induced transactivation of EGFR and cell proliferation, possibly through its interaction with SHP-2 and EGFR. Although tyrosine kinases are critically involved in the angiotensin II (Ang II) type 1 (AT1) receptor signaling, how AT1 receptors activate tyrosine kinases is not fully understood. We examined the structural requirements of the AT1 receptor for transactivation of the epidermal growth factor (EGF) receptor (EGFR). Studies using carboxyl terminal-truncated AT1 receptors indicated that the amino acid sequence between 312 and 337 is required for activation of EGFR. The role of the conserved YIPP motif in this sequence in transactivation of EGFR was investigated by mutating tyrosine 319. Ang II failed to activate EGFR in cells expressing AT1-Y319F, whereas EGFR was activated even without Ang II in cells expressing AT1-Y319E, which mimics the AT1 receptor phosphorylated at Tyr-319. Immunoblot analyses using anti-phospho Tyr-319-specific antibody showed that Ang II increased phosphorylation of Tyr-319. EGFR interacted with the AT1 receptor but not with AT1-Y319F in response to Ang II stimulation, whereas the EGFR-AT1 receptor interaction was inhibited in the presence of dominant negative SHP-2. The requirement of Tyr-319 seems specific for EGFR because Ang II-induced activation of other tyrosine kinases, including Src and JAK2, was preserved in cells expressing AT1-Y319F. Extracellular signal-regulated kinase activation was also maintained in AT1-Y319F through activation of Src. Overexpression of wild type AT1 receptor in cardiac fibroblasts enhanced Ang II-induced proliferation. By contrast, overexpression of AT1-Y319F failed to enhance cell proliferation. In summary, Tyr-319 of the AT1 receptor is phosphorylated in response to Ang II and plays a key role in mediating Ang II-induced transactivation of EGFR and cell proliferation, possibly through its interaction with SHP-2 and EGFR. The signaling mechanism of the angiotensin II (Ang II) 1The abbreviations used are: Ang II, angiotensin II; AT1, Ang II type 1; EGF, epidermal growth factor; EGFR, EGF receptor; C-tail, carboxyl terminus; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; WT, wild type; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; IPx, inositol phosphates type 1 (AT1) receptor has traditionally been portrayed to be dependent on heterotrimeric G proteins, including Gαq and Gαi proteins and their downstream targets, primarily phospholipase C (1Griendling K.K. Lassegue B. Murphy T.J. Alexander R.W. Adv. Pharmacol. 1994; 28: 269-306Google Scholar). This results in inositol triphosphate generation, which in turn causes an increase in intracellular calcium concentrations and diacylglycerol formation, leading to activation of protein kinase C. However, recent investigations revealed that tyrosine phosphorylation is also intimately involved in AT1 receptor signaling (2Berk B.C. Corson M.A. Circ. Res. 1997; 80: 607-616Google Scholar, 3Bernstein K.E. Ali M.S. Sayeski P.P. Semeniuk D. Marrero M.B. Lab. Invest. 1998; 78: 3-7Google Scholar, 4Du J. Sperling L.S. Marrero M.B. Phillips L. Delafontaine P. Biochem. Biophys. Res. Commun. 1996; 218: 934-939Google Scholar, 5Leduc I. Haddad P. Giasson E. Meloche S. Mol. Pharmacol. 1995; 48: 582-592Google Scholar, 6Sadoshima J. Circ. Res. 1998; 82: 1352-1355Google Scholar). Ang II-induced ERK1/2 activation, for example, requires tyrosine kinase activation, including Src family tyrosine kinases (7Sadoshima J. Izumo S. EMBO J. 1996; 15: 775-787Google Scholar,8Ishida M. Ishida T. Thomas S. Berk B.C. Circ. Res. 1998; 1998: 7-12Google Scholar) and epidermal growth factor receptor (EGFR) (9Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Google Scholar, 10Murasawa S. Mori Y. Nozawa Y. Gotoh N. Shibuya M. Masaki H. Maruyama K. Tsutsumi Y. Moriguchi Y. Shibazaki Y. Tanaka Y. Iwasaka T. Inada M. Matsubara H. Circ. Res. 1998; 82: 1338-1348Google Scholar). It is unclear, however, how AT1 receptors, which lack intrinsic tyrosine kinase activities, are able to stimulate tyrosine kinases. We have recently shown that an AT1 receptor second intracellular loop mutant, lacking heterotrimeric G protein coupling, is able to activate Src tyrosine kinase (11Seta K. Nanamori M. Modrall J.G. Neubig R.R. Sadoshima J. J. Biol. Chem. 2002; 277: 9268-9277Google Scholar). This suggests that heterotrimeric G protein-independent mechanisms are able to activate Src. Furthermore, increasing lines of evidence suggest that the carboxyl terminus (C-tail) of the AT1 receptor plays an important role in the AT1 receptor signaling (11Seta K. Nanamori M. Modrall J.G. Neubig R.R. Sadoshima J. J. Biol. Chem. 2002; 277: 9268-9277Google Scholar, 12Franzoni L. Nicastro G. Pertinhez T.A. Tato M. Nakaie C.R. Paiva A. Schreier S. Spisni A. J. Biol. Chem. 1997; 272: 9734-9741Google Scholar). For example, ligand binding to the AT1 receptor induces physical association of the C-tail of the AT1 receptor with Jak2, thereby causing phosphorylation and translocation of STAT to the nucleus (13Ali M.S. Sayeski P.P. Dirksen L.B. Hayzer D.J. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1997; 272: 23382-23388Google Scholar). Other signaling molecules, including phospholipase Cγ and SHP-2, also have been shown to interact with the C-tail of the AT1 receptor (14Venema R.C. Ju H. Venema V.J. Schieffer B. Harp J.B. Ling B.N. Eaton D.C. Marrero M.B. J. Biol. Chem. 1998; 273: 7703-7708Google Scholar, 15Marrero M.B. Venema V.J. Ju H. Eaton D.C. Venema R.C. Am. J. Physiol. 1998; 275: C1216-C1223Google Scholar). These results suggest that direct interaction between the heterotrimeric G protein-coupled receptor and intracellular signaling molecules may play an important role in mediating activation of downstream-signaling mechanisms. Accumulating data suggests that EGFR is involved in signal transduction of many G protein-coupled receptors, including the AT1 receptor (9Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Google Scholar, 10Murasawa S. Mori Y. Nozawa Y. Gotoh N. Shibuya M. Masaki H. Maruyama K. Tsutsumi Y. Moriguchi Y. Shibazaki Y. Tanaka Y. Iwasaka T. Inada M. Matsubara H. Circ. Res. 1998; 82: 1338-1348Google Scholar) (for review, see Ref. 16Ullian M.E. Linas S.L. J. Clin. Invest. 1989; 84: 840-846Google Scholar). Ang II induces tyrosine phosphorylation of EGFR and its association with Shc and Grb2, leading to subsequent activation of the Ras-Raf-MEK-ERK1/2 pathway (9Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Google Scholar). Although several signaling mechanisms are involved in Ang II-induced activation of EGFR (9Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Google Scholar, 17Sadoshima J. Izumo S. Circ. Res. 1993; 73: 413-423Google Scholar, 18Iwaki K. Sukhatme V.P. Shubeita H.E. Chien K.R. J. Biol. Chem. 1990; 265: 13809-13817Google Scholar, 19Berridge M.J. Downes C.P. Hanley M.R. Biochem. J. 1982; 206: 587-595Google Scholar, 20Aoki H. Richmond M. Izumo S. Sadoshima J. Biochem. J. 2000; 347: 275-284Google Scholar), whether or not direct interaction between the AT1 receptor and intracellular signaling molecules is required for EGFR activation and, if so, the amino acid sequence of the AT1 receptor mediating Ang II-induced EGFR activation has not been identified. To elucidate the molecular mechanism of Ang II-induced EGFR activation, we investigated the structural requirements of the AT1 receptor and the associating signaling mechanism leading to transactivation of EGFR. Our results indicate that tyrosine 319 at the conserved YIPP motif in the carboxyl terminus of the AT1 receptor plays an essential role in mediating Ang II-induced transactivation of EGFR and cell proliferation. Ang II was purchased from Peninsula. Anti-FLAG M2 affinity gel was from Sigma. Horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody (RC20H) and anti-EGF receptor monoclonal antibody were from Transduction Laboratories. Anti-v-Src monoclonal antibody was from Calbiochem. Anti-EGF receptor sheep polyclonal antibody was from Upstate Biotechnology. Rabbit anti-ERK1/2 polyclonal antibody was from Zymed Laboratories Inc., and rabbit anti-active ERK1/2 polyclonal antibody was from Promega. Horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG antibodies were from Cell Signaling Technology. Anti-AT1 receptor antibody was from Santa Cruz Biotechnology. Dowex AG1-X8 formate resin was from Bio-Rad. 3-[4-Iodotyrosyl-125I]Ang II and myo-[3H]inositol were from Amersham Biosciences. Enolase was from Roche Molecular Biochemicals. AG1478 was from Biomol. The full-length rat AT1a wild type receptor (AT1-WT) cDNA subcloned into pcDM8 was obtained from Dr. J. Harrison. The following AT1a receptor mutants were generated by using PCR and QuikChange (Stratagene): AT1-Y319F, tyrosine 319 was replaced with phenylalanine; AT1-Y319E, tyrosine 319 was replaced with glutamic acid; AT1-(1–338), the carboxyl terminus of the AT1 receptor (339–359) was truncated; AT1-(1–311), the carboxyl terminus of the AT1 receptor (312–359) was truncated. Mammalian expression plasmid encoding the AT1 receptor carboxyl terminus peptide was generated by subcloning cDNA encoding AT1-(292–359) into a mammalian expression vector pHM6 (Roche Molecular Biochemicals), which has an HA tag in the amino terminus. Site-directed mutagenesis was performed to generate HA-AT1-(292–359)-Y319F using QuikChange. The DNA sequence of all constructs was confirmed by DNA sequence analyses. Expression plasmids for EGFR and JAK2 were provided by Dr. A. Yoshimura (Kyushu University, Fukuoka, Japan). Plasmid for Myc-SHP-2 was provided by Dr. S. Sano (Mitsubishi Kasei Institute of Life Science, Tokyo, Japan). Plasmid encoding Myc-dominant negative SHP-2 (SHP-2-(1–225)) was generated by PCR, and the PCR product was subjected to TA cloning using pCR3.1 vector (Invitrogen). Plasmid encoding dominant negative Src (K296R/Y528F) and its control vector (pUSE) were purchased from Upstate Biotechnology. COS-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin at 37 °C in a humidified 5% CO2 atmosphere. Transfections were performed on 70% confluent monolayers in 60-mm dishes for immunoprecipitation and Src kinase assays or in 35-mm dishes for ERK1/2 assays. For transient transfection, 2.5 ml of Opti-MEM I (Invitrogen) containing 4 μg of DNA, 8 μl of LipofectAMINE Plus reagent, and 12 μl of LipofectAMINE was used for a 60-mm dish. One ml of Opti-MEM I containing 2 μg of DNA, 6 μl of LipofectAMINE Plus reagent, and 4 μl of LipofectAMINE was used for a 35-mm dish. Empty pcDNA 3.0 plasmid was added as needed to keep the total amount of DNA constant per transfection. Cells were incubated in serum-free Opti-MEM at 37 °C for 3 h. COS-7 cells were then incubated with 10% fetal bovine serum in DMEM and incubated overnight. Transfected cells were serum-starved in serum-free DMEM for 24–36 h before stimulation. Assays were performed 48 h after transfection. Saturation binding curves were determined using a modification of the whole cell receptor binding assay (16Ullian M.E. Linas S.L. J. Clin. Invest. 1989; 84: 840-846Google Scholar) as described previously (11Seta K. Nanamori M. Modrall J.G. Neubig R.R. Sadoshima J. J. Biol. Chem. 2002; 277: 9268-9277Google Scholar). The B max and the dissociation constant (K d) 3-[4-iodotyrosyl-125I]AngII binding was determined by using Prism 3.0 (GraphPad). Protein assay was performed on each sample using the Bio-Rad protein assay kit. Cardiac fibroblast cultures were prepared as described previously (17Sadoshima J. Izumo S. Circ. Res. 1993; 73: 413-423Google Scholar). In brief, hearts were removed from 1-day-old Crl:(WI)BR-Wistar rats (Charles River Laboratories) and subjected to digestion with collagenase type IV (Sigma), 0.1% trypsin (Invitrogen), and 15 μg/ml DNase I (Sigma). Cell suspensions were applied on a discontinuous Percoll gradient (1.060/1.086 g/ml) and subjected to centrifugation at 3000 rpm for 30 min (18Iwaki K. Sukhatme V.P. Shubeita H.E. Chien K.R. J. Biol. Chem. 1990; 265: 13809-13817Google Scholar). The layer containing primarily non-cardiac myocytes was removed and subjected to the preplating procedure for 1 h. The supernatant was discarded, and the attached cells were cultured in the media containing DMEM/F-12 supplemented with 10% fetal bovine serum. Cells were passed twice to enrich for cardiac fibroblasts. Cells were cultured in serum-free conditions for 48 h before experiments. Cell stimulation was carried out at 37 °C in serum-free medium. After stimulation, COS-7 cells were scraped and lysed in CHAPS buffer (150 mm NaCl, 40 mm HCl-Tris, pH 7.5, 1% Triton X-100, 0.1% CHAPS, 10% glycerol, 2 mm EDTA, 0.1 mm NaVO4, 1 mm NaF, 0.5 mm4-(2-aminoethyl)benzenesulfonyl fluoride, 0.5 μg/ml aprotinin, 0.5 μg/ml leupeptin) for immunoprecipitation or hypoosmotic lysis buffer (25 mm NaCl, 25 mm Tris, pH 7.5, 0.5 mm EGTA, 10 mm sodium pyrophosphate, 1 mm Na3VO4, 10 mm NaF, 0.5 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.5 μg/ml aprotinin, 0.5 μg/ml leupeptin) for ERK1/2 kinase assays. Cell lysates were incubated on ice for 10 min and subjected to centrifugation for 30 min. Protein concentrations of the supernatants were adjusted to be 1 mg/ml with the lysis buffer. For immunoprecipitation of EGFR-FLAG, the cell lysates (500 μg) were incubated with 40 μl of anti-FLAG M2 affinity gel at 4 °C for 2 h. For immunoprecipitation of endogenous EGFR, the cell lysates were incubated with 4 μg of anti-EGFR monoclonal antibody for 1 h followed by protein G-agarose (30 μl of slurry) for 45 min. The immune complexes were washed 3 times with lysis buffer and denatured in Laemmli sample buffer. After SDS-PAGE, samples were transferred onto polyvinylidene fluoride microporous membranes (Millipore). Immunoblots were performed as described previously. Phosphorylated levels of the EGFR, the AT1 receptor, or ERK1/2 were analyzed by immunoblotting with anti-phosphospecific antibody and scanning densitometry, and the results were expressed as fold increase compared with the control. Polyclonal anti-phospho-specific antibody was generated by injecting Ac-QLLK(pY)IPPKAKS(Ahx)C-amide (pY is phosphotyrosine; Ahx, is a six-carbon spacer) into rabbits as an immunogen. Generated antibody was then subjected to affinity purification (BIOSOURCE International). Measurement of inositol phosphates (IPx) was based upon the method of Berridgeet al. (19Berridge M.J. Downes C.P. Hanley M.R. Biochem. J. 1982; 206: 587-595Google Scholar) as described previously (11Seta K. Nanamori M. Modrall J.G. Neubig R.R. Sadoshima J. J. Biol. Chem. 2002; 277: 9268-9277Google Scholar). Cells were incubated with myo-[3H]inositol (10 μCi/ml) in DMEM for 24 h at 37 °C. Labeling was terminated by aspirating the medium, rinsing cells with oxygenated reaction buffer (142 mm NaCl, 30 mm Hepes buffer, pH 7.4, 5.6 mm KCl, 3.6 mm NaHCO3, 2.2 mm CaCl2, 1.0 mm MgCl2, and 1 mg/ml d-glucose), and harvesting cells with phosphate-buffered saline, 0.02%EDTA. Cells were centrifuged twice (300 × g, 5 min) in reaction buffer, and the pellet was resuspended in an equal volume of reaction buffer containing 60 mm LiCl. Stimulation of IPx production was initiated by mixing 0.25 ml of cell suspension with 0.25 ml of 0–100 nmAng II in reaction buffer (without LiCl). The mixture was incubated for 30 min at 37 °C, then 0.5 ml of ice-cold 20% trichloroacetic acid was added. Precipitates were pelleted (4100 × g, 20 min), and the trichloroacetic acid-soluble fraction was transferred to new tubes, washed with water-saturated diethyl ether, and neutralized with NaHCO3. IPx were isolated by adsorption to 0.5 ml of Dowex AG1-X8 formate resin slurry and rinsed 5 times with 3 ml of unlabeled 5 mm myoinositol followed by elution with 1 ml of 1.2 m ammonium formate, 0.1 m formic acid. The elutes were counted by liquid scintillation counter in 5 ml of ScintiVerse. The tyrosine kinase activity of Src was determined by the immune complex kinase assay using enolase as a substrate as described previously (7Sadoshima J. Izumo S. EMBO J. 1996; 15: 775-787Google Scholar, 11Seta K. Nanamori M. Modrall J.G. Neubig R.R. Sadoshima J. J. Biol. Chem. 2002; 277: 9268-9277Google Scholar). Cell lysates were prepared in a lysis buffer (150 mm NaCl, 15 mm HEPES, pH 7.0, 1% deoxycholic acid, 1% IGEPAL, 0.1% SDS, 0.1 mmNaVO4, 1 mm NaF, 0.5 mm4-(2-aminoethyl)benzenesulfonyl fluoride, 0.5 μg/ml aprotinin, 0.5 μg/ml leupeptin). The cell lysates containing equal amount of protein (750 μg) were incubated with anti-v-Src monoclonal antibody at 4 °C for 1 h. Protein G-Sepharose was then added. The immunoprecipitates were washed twice with lysis buffer without SDS or deoxycholic acid and then washed once with kinase buffer (50 mm HEPES, pH 7.6, 0.1 mm EDTA, 10 mm MnCl2, 0.015% Brig 35). Pellets were incubated for 15 min at 37 °C in the kinase buffer with 1 μCi of [γ-32P]ATP and 0.25 μg of enolase as a substrate. The reaction was terminated by the addition of Laemmli sample buffer on ice. Reaction mixtures were boiled and subjected to 12% SDS-PAGE followed by autoradiography. Results were analyzed by densitometry. Adenovirus-mediated transduction was performed as described previously (20Aoki H. Richmond M. Izumo S. Sadoshima J. Biochem. J. 2000; 347: 275-284Google Scholar). Cells grown in 60-mm dishes were transduced with an adenovirus vector harboring dominant-negative Ras (Ad5.N17Ras) (courtesy of Dr. M. Schneider, Baylor College of Medicine, Houston, TX) at a multiplicity of infection of 100. For a control study, Ad5/ΔE1sp1B (courtesy of Dr. B. French, University of Virginia, Charlottesville, VA) was used. Adenovirus vectors harboring the wild type AT1 receptor or AT1-Y319F were generated by using the AdEasy system (21He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Google Scholar). All experiments were performed 48 h after transduction. Cells were plated at a density of 0.3 × 106/well in six-well plates. Twelve hours after plating, cells were serum-starved for 12 h and then stimulated with Ang II (10−7m) in the presence or absence of AG1478 (250 nm) for 36 h. Ang II or AG1478 was added every 12 h. After stimulation, cells were washed twice with phosphate-buffered saline. The cell layer was scraped with 1 ml of standard sodium citrate containing 0.25% SDS and vortexed extensively. Total DNA content was determined by the Hoechst dye method as described previously (17Sadoshima J. Izumo S. Circ. Res. 1993; 73: 413-423Google Scholar). Data are given as the mean ± S.E. Statistical analyses were performed using the analysis of variance. The post-test comparison was performed by the method of Tukey. Significance was accepted at p < 0.05 level. Ang II stimulation of COS-7 cells without transfection of the AT1 receptor did not activate either endogenous or transfected EGFRs (not shown). By contrast, in COS-7 cells transfected with the AT1 receptor, Ang II, caused time-dependent increases in tyrosine phosphorylation of either endogenous (not shown) or transfected EGFRs (Fig. 1 A). Tyrosine phosphorylation of EGFR by Ang II was observed within 3 min, reached a peak around 5 min, and lasted for more than 60 min (Fig.1 B (n = 7) and 2.8 ± 0.1-fold at 60 min (n = 3)). This suggests that stimulation of the AT1 receptor causes transactivation of EGFR in COS-7 cells. Because it has been suggested that the C-tail of the AT1 receptor plays an important role in mediating cell-signaling mechanisms of the AT1 receptor (11Seta K. Nanamori M. Modrall J.G. Neubig R.R. Sadoshima J. J. Biol. Chem. 2002; 277: 9268-9277Google Scholar), we examined the role of the AT1 receptor C-tail in Ang II-induced transactivation of EGFR. We co-transfected EGFR-FLAG and carboxyl-terminal-truncated AT1 receptors into COS-7 cells, and EGFR was immunoprecipitated with anti-FLAG antibody. Although Ang II caused significant increases in tyrosine phosphorylation of EGFR in cells transfected with AT1-(1–338), it failed to do so in cells transfected with AT1-(1–311) (Fig. 2 A). These results suggest that the amino acid sequence located between amino acid 312 and 337 of the AT1 receptor is required for activation of EGFR by Ang II stimulation. Between amino acids 312 and 337 of the AT1 receptor, the YIPP-(319–322) motif is evolutionarily conserved in AT1 receptors cloned from many species (Fig. 2 B). It has been shown that several signaling molecules directly or indirectly associate with the YIPP motif in the AT1 receptor (13Ali M.S. Sayeski P.P. Dirksen L.B. Hayzer D.J. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1997; 272: 23382-23388Google Scholar, 14Venema R.C. Ju H. Venema V.J. Schieffer B. Harp J.B. Ling B.N. Eaton D.C. Marrero M.B. J. Biol. Chem. 1998; 273: 7703-7708Google Scholar). To test the role of this conserved motif in Ang II-induced EGFR activation, we made a mutant where a tyrosine residue at position 319 in this motif was mutated to phenylalanine (AT1-Y319F). Interestingly, Ang II-induced activation of EGFR was abolished in COS-7 cells transfected with AT1-Y319F (Fig.2 C). To test if phosphorylation of tyrosine 319 is involved in activation of EGFR, we mutated tyrosine 319 to glutamate (Y319E) to mimic the status of phosphorylation. Expression of AT1-Y319E in COS-7 cells significantly increased phosphorylation of EGFR in basal conditions, and Ang II stimulation failed to increase phosphorylation of EGFR (Fig. 3 A). These results suggest that phosphorylation of tyrosine 319 may be involved in Ang II-induced activation of EGFR. To test if tyrosine 319 of the AT1 receptor is phosphorylated in vivo in response to Ang II stimulation, we generated phosphotyrosine 319-specific anti-AT1 receptor antibody (anti-phosphotyrosine 319 antibody). Either wild type AT1 receptor (AT1-WT) or AT1-Y319F was transfected in COS-7 cells. The AT1 receptor phosphorylated at tyrosine 319 was immunoprecipitated by the anti-phosphotyrosine 319 antibody and immunoblotted with the same antibody. Although AT1-WT was not detected by anti-phosphotyrosine 319 antibody in unstimulated cells, it was detected after the cells were stimulated with Ang II for 3 min (Fig. 3 B). Phosphorylation of tyrosine 319 was transient and returned to the basal level at 5 min. No apparent signals of phosphotyrosine 319 were detected in samples obtained from cells expressing AT1-Y319F (Fig. 3 B), suggesting that the signal found in AT1-WT was most likely from phosphorylated Tyr-319. Duplicate samples were subjected to immunoprecipitation using anti-(total)-AT1 receptor antibody and immunoblotted with the same antibody. The result confirmed that similar amounts of the AT1 receptor were solubilized in each sample (Fig.3 C). These results indicate that tyrosine 319 in AT1-WT is transiently phosphorylated in response to Ang II stimulation. To confirm that tyrosine 319 of the AT1 receptor is important for Ang II-induced transactivation of EGFR, we examined the effect of overexpression of a mini-gene-containing AT1 receptor carboxyl terminus peptide (HA-AT1-C) upon Ang II-induced EGFR activation in COS-7 cells. Expression of HA-AT1-C inhibited Ang II-induced activation of EGFR in COS-7 cells (Fig. 4 A). Overexpression of HA-AT1-C alone did not inhibit activation of EGFR by EGF treatment (Fig. 4 B), suggesting that the effect of HA-AT1-C is stimulus-specific. Furthermore, a mini-gene-containing HA-AT1-C, whose tyrosine 319 is mutated to phenylalanine (HA-AT1-C-Y319F), failed to inhibit Ang II-induced EGFR activation in COS-7 cells (Fig.4 A). These results further support the notion that tyrosine 319 in the AT1 receptor plays an essential role in mediating EGFR activation by the AT1 receptor. We next examined if the AT1 receptor and EGFR physically associate with each other. We co-transfected AT1 receptors and EGFR into COS-7 cells and stimulated the cells with Ang II. EGFR immunoprecipitates were immunoblotted with anti-AT1 receptor antibody. Interestingly, AT1 receptors were co-immunoprecipitated with EGFR in samples stimulated with Ang II for 3 min (2.7 ± 0.2-fold versus 0 min,n = 4, Fig.5 A). This interaction between the AT1 receptor and EGFR was transient, since it was not observed at 5 min. Equal amounts of EGFRs were immunoprecipitated in each sample, and the immunoprecipitated EGFR was tyrosine-phosphorylated in response to Ang II stimulation, consistent with the results shown in Fig. 1 (Fig.5 A). Because it has been shown that SHP-2 is associated with AT1 receptor at the YIPP motif (14Venema R.C. Ju H. Venema V.J. Schieffer B. Harp J.B. Ling B.N. Eaton D.C. Marrero M.B. J. Biol. Chem. 1998; 273: 7703-7708Google Scholar, 15Marrero M.B. Venema V.J. Ju H. Eaton D.C. Venema R.C. Am. J. Physiol. 1998; 275: C1216-C1223Google Scholar), we examined if disrupting interaction between the AT1 receptor and SHP-2 affects interaction between the AT1 receptor and EGFR and abolishes Ang II-induced activation of EGFR. Ang II-dependent interaction between the AT1 receptor and EGFR and activation of EGFR were both inhibited in the presence of dominant negative SHP-2 (22Kim H. Baumann H. Mol. Cell. Biol. 1999; 19: 5326-5338Google Scholar) (Fig. 5 A). These results suggest that SHP-2 plays an essential role in mediating Ang II-induced activation of EGFR. Because tyrosine 319 plays an essential role in mediating Ang II-induced activation of EGFR, we attempted to determine if EGFR can interact with AT1-Y319F. EGFR and AT-Y319F were co-transfected into COS-7 cells, and cells were stimulated with Ang II. EGFR was immunoprecipitated and then immunoblotted with anti-AT1 receptor antibody. We did not detect the AT1 receptor in the EGFR immunoprecipitates (Fig. 5 B). As expected, EGFR was not phosphorylated in response to Ang II stimulation in these experiments. These results suggest that tyrosine 319 plays an essential role in mediating Ang II-dependent interaction between the AT1 receptor and EGFR. We examined if other signaling mechanisms are also affected in the AT1-Y319F mutant. Ang II caused significant levels of IPx accumulation in cells transfected with AT1-Y319F, which was not statistically different from those in cells transfected with AT1-WT (Fig. 6 A). This suggests that AT1-Y319F maintains coupling with the Gαq-phospholipase C pathway. This also suggests that activation of IPx alone is not sufficient for the AT1 receptor to mediate EGFR activation. We attempted to determine if activation of other tyrosine kinases is also affected in AT1-Y319F. Ang II caused significant increases in Src activities in cells transfected with either AT1-WT or AT1-Y319F (2.9 ± 0.1-fold in WT, 2.7 ± 0.2-fold in Y319F at 5 min) (Fig. 6 B). Ang II also caused similar levels of increases in tyrosine phosphorylation of transfected JAK2-FLAG in cells expressing either AT1-WT or AT1-Y319F (3.5 ± 0.1-fold in WT, 3.4 ± 0.2-fold in Y319F at 5 min) (Fig. 6 C). These results suggest that Ang II-induced activation of some tyrosine kinases, including Src and JAK2, is preserved in cells expressing AT1-Y319F. Ang II activated ERKs (3.5 ± 0.1-fold,p < 0.05 versus control) in COS-7 cells transfected with AT1-WT (Fig.7 A). AG1478, a specific inhibitor for EGFR, abolished Ang II-induced ERK activation in COS-7 cells transfected with AT1-WT (Fig. 7 A), suggesting that EGFR plays an essential role in ERK activation by AT1-WT in COS-7 cells. Surprisingly, however, Ang II was still able to activate ERKs in AT1-Y319F (3.6 ± 0.2-fold, p < 0.05versus control), where Ang II-induc" @default.
• W1993265529 created "2016-06-24" @default.
• W1993265529 creator A5067329924 @default.
• W1993265529 creator A5091471046 @default.
• W1993265529 date "2003-03-01" @default.
• W1993265529 modified "2023-10-14" @default.
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