Koichi Seta

Inactivation of glycogen synthase kinase 3beta (GSK3beta) is critical for transcription of atrial natriuretic factor (ANF) by beta-adrenergic receptors in cardiac myocytes. We examined the mechanism by which GSK3beta regulates ANF transcription. Stimulation of beta-adrenergic receptors induced nuclear accumulation of GATA4, whereas beta-adrenergic ANF transcription was suppressed by dominant negative GATA4, suggesting that GATA4 plays an important role in beta-adrenergic ANF transcription. Interestingly, GATA4-mediated transcription was markedly attenuated by GSK3beta. GSK3beta physically associates with GATA4 and phosphorylates GATA4 in vitro. Overexpression of GSK3beta suppressed both basal and beta-adrenergic increases in nuclear expression of GATA4, whereas inhibition of GSK3beta by LiCl caused nuclear accumulation of GATA4, suggesting that GSK3beta negatively regulates nuclear expression of GATA4. The nuclear exportin Crm1 reduced nuclear expression of GATA4, and the reduction was enhanced by GSK3beta but not by kinase-inactive GSK3beta. Leptomycin B, an inhibitor for Crm1, increased basal nuclear GATA4 and suppressed GSK3beta-induced decreases in nuclear GATA4. These results suggest that GSK3beta negatively regulates nuclear expression of GATA4 by stimulating Crm1-dependent nuclear export. Inhibition of GSK3beta by beta-adrenergic stimulation abrogates GSK3beta-induced nuclear export of GATA4, causing nuclear accumulation of GATA4, which may represent an important signaling mechanism mediating cardiac hypertrophy.

Angiotensin II (Ang II) type 1 receptors (AT1Rs) activate tyrosine kinases, including Src. Whether or not tyrosine kinase activation by AT1R occurs independently of heterotrimeric G protein coupling and, if so, the cellular function of such a mechanism are unknown. To address these questions, we used an AT1aR intracellular second loop mutant, which lacks heterotrimeric G protein coupling (AT1a-i2m). Surprisingly, Ang II-induced Src activation was preserved in AT1a-i2m, which was not attenuated by inhibiting protein kinase C and Ca(2+) or by inhibiting Galpha(i) or Galpha(q) in CHO-K1 cells. By contrast, Ang II-induced Src activation was abolished in a C-terminally truncated AT1a-(1--309), where Ang II-induced inositol phosphate response was preserved. Ang II activates ERKs via a Src-Ras-dependent mechanism in AT1a-i2m. ERKs activated by AT1a-i2m phosphorylate their cytoplasmic targets, including p90(RSK), but fail to translocate into the nucleus or to cause cell proliferation. Ang II-induced nuclear translocation of ERKs by wild type AT1aR was inhibited by overexpression of nuclear exportin Crm-1, while that by AT1a-i2m was restored by leptomycin B, an inhibitor of Crm-1. In summary, while Src and ERKs are activated by Ang II even without heterotrimeric G protein coupling, the carboxyl terminus of the AT1 receptor is required for activation of Src. Interestingly, ERKs activated by heterotrimeric G protein-independent mechanisms fail to phosphorylate nuclear targets due to lack of inhibition of Crm-1-induced nuclear export of ERKs. These results suggest that heterotrimeric G protein-dependent and -independent signaling mechanisms play distinct roles in Ang II-mediated cellular responses.

Background Arterial stiffness is an important predictor of cardiovascular events; however, indexes for measuring arterial stiffness have not been widely incorporated into routine clinical practice. This study aimed to determine whether the cardio‐ankle vascular index (CAVI), based on the blood pressure–independent stiffness parameter β and reflecting arterial stiffness from the origin of the ascending aorta, is a good predictor of cardiovascular events in patients with cardiovascular disease risk factors in a large prospective cohort. Methods and Results This multicenter prospective cohort study, commencing in May 2013, with a 5‐year follow‐up period, included patients (aged 40‒74 years) with cardiovascular disease risks. The primary outcome was the composite of cardiovascular death, nonfatal stroke, or nonfatal myocardial infarction. Among 2932 included patients, 2001 (68.3%) were men; the mean (SD) age at diagnosis was 63 (8) years. During the median follow‐up of 4.9 years, 82 participants experienced primary outcomes. The CAVI predicted the primary outcome (hazard ratio, 1.38; 95% CI, 1.16‒1.65; P <0.001). In terms of event subtypes, the CAVI was associated with cardiovascular death and stroke but not with myocardial infarction. When the CAVI was incorporated into a model with known cardiovascular disease risks for predicting cardiovascular events, the global χ 2 value increased from 33.8 to 45.2 ( P <0.001), and the net reclassification index was 0.254 ( P =0.024). Conclusions This large cohort study demonstrated that the CAVI predicted cardiovascular events. Registration URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01859897.

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. monoclonal antibody was from receptor antibody was from antibody was from and ERK1/2 antibody was from Horseradish peroxidase-conjugated and were from receptor antibody was from was from II and were from was from was from The wild type receptor was from J. The receptor were by using and AT1-Y319F, tyrosine 319 was with AT1-Y319E, tyrosine 319 was with acid; the carboxyl terminus of the AT1 receptor was the carboxyl terminus of the AT1 receptor was the AT1 receptor carboxyl terminus was by a which has an in the amino was to using The sequence of was by sequence for EGFR and were by A. for was by S. of negative SHP-2 was by and the was to using dominant negative Src and its were purchased from cells were maintained in Dulbecco's modified Eagle's with and at in a were on in for and Src kinase or in for ERK1/2 For of of of and of was used for a of of of and of was used for a was to the of were in at for cells were with in and cells were in for were binding were using a of the cell receptor binding M.E. Linas S.L. J. Clin. Invest. 1989; 84: 840-846Google Scholar) (11Seta K. Nanamori M. Modrall J.G. Neubig R.R. Sadoshima J. J. Biol. Chem. 2002; 277: 9268-9277Google Scholar). The and the binding was by using was on using the protein were J. Izumo S. Circ. Res. 1993; 73: 413-423Google Scholar). In were from and to with type and were on a and to at for K. Sukhatme V.P. Shubeita H.E. Chien K.R. J. Biol. Chem. 1990; 265: 13809-13817Google Scholar). The primarily was and to the for 1 The was and the cells were in the with were to for cardiac were in for was at in stimulation, cells were and in CHAPS, 1 for or 1 for ERK1/2 kinase were on for and to for concentrations of the were to be 1 with the For of the cell were with of M2 affinity gel at for For of EGFR, the cell were with of monoclonal antibody for 1 by protein of for The were with and in were were of the EGFR, the AT1 or ERK1/2 were by with antibody and and the results were increase with the antibody was by is is a an antibody was to affinity of inositol phosphates was the of M.J. Downes C.P. Hanley M.R. Biochem. J. 1982; 206: 587-595Google Scholar) (11Seta K. Nanamori M. Modrall J.G. Neubig R.R. Sadoshima J. J. Biol. Chem. 2002; 277: 9268-9277Google Scholar). were with in for at was by the cells with and 1 and cells with were in and the was in an of of was by of cell with of II in The was for at of acid was were and the was to with and with were by to of and with of by with 1 of The were by in of The tyrosine kinase of Src was by the kinase using a (7Sadoshima J. Izumo S. EMBO J. 1996; 15: 775-787Google Scholar, K. Nanamori M. Modrall J.G. Neubig R.R. Sadoshima J. J. Biol. Chem. 2002; 277: 9268-9277Google Scholar). were in a 1 The cell of protein were with monoclonal antibody at for 1 was The were with without or acid and with kinase were for at in the kinase with 1 of and of a The was by the of on were and to by were by transduction was H. Richmond M. Izumo S. Sadoshima J. Biochem. J. 2000; 347: 275-284Google Scholar). in were with an of M. of at a of of For a of B. of was the wild type AT1 receptor or AT1-Y319F were by using the S. J. B. S. A. 1998; Scholar). were were at a of in cells were for and with Ang II in the presence or of for Ang II or was stimulation, cells were with The cell was with 1 of and was by the J. Izumo S. Circ. Res. 1993; 73: 413-423Google Scholar). are the analyses were using the of The was by the of was at Ang II of cells without of the AT1 receptor not activate or By contrast, in cells with the AT1 Ang II, in tyrosine phosphorylation of or 1 phosphorylation of EGFR by Ang II was a and for and at This suggests that of the AT1 receptor causes transactivation of EGFR in has been that the C-tail of the AT1 receptor plays an important role in mediating 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 and AT1 receptors and EGFR was with Although Ang II in tyrosine phosphorylation of EGFR in cells with failed to in cells with These results suggest that the amino acid sequence between amino acid 312 and 337 of the AT1 receptor is required for activation of EGFR by Ang II amino 312 and 337 of the AT1 the motif is conserved in AT1 receptors from many It has been shown that several signaling molecules or 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, 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 the role of this conserved motif in Ang II-induced EGFR activation, we a a tyrosine at 319 in this motif was to Ang II-induced activation of EGFR was in cells with AT1-Y319F To if phosphorylation of tyrosine 319 is involved in activation of EGFR, we tyrosine 319 to to the of of in cells increased phosphorylation of EGFR in and Ang II failed to increase phosphorylation of EGFR These results suggest that phosphorylation of tyrosine 319 may be involved in Ang II-induced activation of EGFR. To if tyrosine 319 of the AT1 receptor is phosphorylated in in response to Ang II stimulation, we receptor antibody 319 wild type AT1 receptor or AT1-Y319F was in The AT1 receptor phosphorylated at tyrosine 319 was by the anti-phosphotyrosine 319 antibody and with the Although was not by anti-phosphotyrosine 319 antibody in was the cells were with Ang II for of tyrosine 319 was and to the at of 319 were in from cells expressing AT1-Y319F that the signal in was from phosphorylated Tyr-319. were to using receptor antibody and with the The that of the AT1 receptor were in These results indicate that tyrosine 319 in is phosphorylated in response to Ang II To that tyrosine 319 of the AT1 receptor is important for Ang II-induced transactivation of EGFR, we examined the of overexpression of a AT1 receptor carboxyl terminus Ang II-induced EGFR activation in of inhibited Ang II-induced activation of EGFR in cells Overexpression of not activation of EGFR by EGF that the of is Furthermore, a tyrosine 319 is to failed to Ang II-induced EGFR activation in cells These results the that tyrosine 319 in the AT1 receptor plays an essential role in mediating EGFR activation by the AT1 We examined if the AT1 receptor and EGFR with We AT1 receptors and EGFR cells and the cells with Ang EGFR were with receptor AT1 receptors were with EGFR in with Ang II for This interaction between the AT1 receptor and EGFR was was not at of were in and the EGFR was in response to Ang II stimulation, with the results shown in 1 has been shown that SHP-2 is 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 interaction between the AT1 receptor and SHP-2 interaction between the AT1 receptor and EGFR and Ang II-induced activation of EGFR. Ang interaction between the AT1 receptor and EGFR and activation of EGFR were inhibited in the presence of dominant negative SHP-2 H. H. Mol. Biol. Scholar) These results suggest that SHP-2 plays an essential role in mediating Ang II-induced activation of EGFR. tyrosine 319 plays an essential role in mediating Ang II-induced activation of EGFR, we to if EGFR interact with AT1-Y319F. EGFR and were and cells were with Ang EGFR was and with receptor We not the AT1 receptor in the EGFR EGFR was not phosphorylated in response to Ang II in These results suggest that tyrosine 319 plays an essential role in mediating Ang interaction between the AT1 receptor and EGFR. We examined if other signaling mechanisms are also in the AT1-Y319F Ang II of in cells with AT1-Y319F, which was not from in cells with This suggests that AT1-Y319F with the C This also suggests that activation of is not for the AT1 receptor to EGFR We to if activation of other tyrosine kinases is also in AT1-Y319F. Ang II in Src in cells with or AT1-Y319F in WT, in at Ang II also of in tyrosine phosphorylation of in cells expressing or AT1-Y319F in WT, in at These results suggest that Ang II-induced activation of tyrosine kinases, including Src and JAK2, is preserved in cells expressing AT1-Y319F. Ang II activated in cells with a specific for EGFR, Ang II-induced activation in cells with that EGFR plays an essential role in activation by in however, Ang II was able to activate in AT1-Y319F Ang II-induced EGFR activation is not Ang II-induced activation in cells with AT1-Y319F activation of by AT1-Y319F was not to activation of EGFR. These results suggest that the AT1 receptor through EGFR, the AT1 receptor mutant, which not activate EGFR, through an EGFR, tyrosine kinase Src also plays an important role in mediating activation in cell To the mechanism by which AT1-Y319F we examined the role of Src in Ang II-induced of dominant negative Src not Ang II-induced activation in cells expressing By contrast, dominant negative Src Ang II-induced activation in cells expressing AT1-Y319F not This suggests that Src the of EGFR activation for Ang II-induced activation by AT1-Y319F. It has been shown that and activation of is by Ang II-induced activation of in cells expressing or AT1-Y319F was in the presence of dominant negative that the mechanisms of activation by and AT1-Y319F are to at the of or of Our results indicated that AT1-Y319F Ang II-induced transactivation of EGFR, whereas activation of if not signaling molecules, including IPx, JAK2, and To if AT1-Y319F has from we or AT1-Y319F in cardiac fibroblasts by an sequence was used a with of of or AT1-Y319F were used for Overexpression of in cardiac fibroblasts increased Ang II-induced EGFR By contrast, overexpression of AT1-Y319F failed to enhance Ang II-induced activation of EGFR, that Tyr-319 plays an essential role in mediating Ang II-induced EGFR activation in cardiac fibroblasts overexpression of the in cardiac fibroblasts enhanced Ang II-induced cell proliferation, which was by the with cells By contrast, overexpression of AT1-Y319F in cell in cardiac fibroblasts To the role of EGFR activation in Ang II-induced cardiac proliferation, we the cells with with the Ang II-induced cell in and cardiac fibroblasts These results suggest that the mechanism by Tyr-319 in the AT1 including activation of EGFR, is required for Ang II-induced cell in cardiac The YIPP motif in the AT1 receptor is conserved in of the AT1 receptor family that this motif is involved in important of the AT1 This motif is also in the growth factor and receptors and is involved in activation of phospholipase Cγ M. C. A. Mol. Biol. 1993; Scholar). Although has been shown that several signaling molecules with the 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, 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, L. K. M. V.J. J. Biol. Chem. Scholar, D. E. M. C. M. G. 1998; Scholar), the specific requirement of tyrosine 319 for activation of downstream protein kinases has not been in Ang II-induced activation of Src and was not in AT1-Y319F, the requirement of tyrosine 319 in the AT1 receptor seems specific for activation of EGFR the activation of tyrosine kinases. The structural requirements of the AT1 receptor in Ang II-induced EGFR have not been It has been shown that the AT1 receptor is by ligand binding (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, H. K.K. Lassegue B. M.S. Alexander R.W. 1994; Scholar). However, the tyrosine phosphorylated by ligand binding has not been in signaling molecules the interact with the YIPP motif of the AT1 has been that tyrosine 319 is phosphorylated (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, 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). By using anti-phosphotyrosine AT1 receptor we that tyrosine 319 is phosphorylated in response to Ang Although has been that phosphorylation of the AT1 receptor may the of downstream signaling molecules of the this has not been In has been recently shown that phosphorylation of the AT1 receptor by G protein-coupled receptor kinase not play an essential role in Ang II-induced cell signaling L. J. Biol. Chem. Scholar). In because of increased of EGFR phosphorylation and because Ang II failed to on EGFR activation by phosphorylation of tyrosine 319 seems to Ang II-induced activation of the EGFR. we not which tyrosine kinase is for phosphorylation of tyrosine 319. mechanisms (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), other tyrosine kinases, Src and S. Iwasaki H. Inagami T. Numaguchi K. Yamakawa T. Motley E.D. Owada K.M. Marumo F. Hirata Y. Scholar, J. M. J. J. Biol. Chem. Scholar, Mol. Pharmacol. 2002; Scholar), (for review, see Ref. G. 2000; Scholar), and M. K.K. L. S. Alexander R.W. Biol. Scholar) have been mechanisms of Ang II-induced activation of EGFR in cell The requirement of tyrosine 319 in the AT1 receptor for EGFR activation in the may not but may requirement for EGFR activation by AT1 of and activation of other kinases Src and by Ang II are preserved in AT1-Y319F, activation of molecules is not for Ang II-induced EGFR lines of evidence suggest that heterotrimeric G protein-coupled receptors interact with intracellular signaling molecules G. Pharmacol. M.J. Pharmacol. Scholar). The but not Ang II-induced activation of EGFR. interaction at the tyrosine 319 may Ang II-induced EGFR It has been shown in that the of with the YIPP Tyr-319 is tyrosine is phosphorylated (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, 15Marrero M.B. Venema V.J. Ju H. Eaton D.C. Venema R.C. Am. J. Physiol. 1998; 275: C1216-C1223Google Scholar). Our results suggest that the AT1 receptor and EGFR interact with other in a ligand and the of their interaction with that of Tyr-319 we that to Tyr-319 Tyr-319 is thereby a dominant negative SHP-2 H. H. Mol. Biol. Scholar) was able to AT1 interaction and Ang II-induced transactivation of EGFR, is that SHP-2 AT1 It has been recently shown that EGFR with receptor in a ligand possibly through a S. S. Y. J. Biol. Chem. 2000; 275: Scholar). SHP-2 and proteins to interaction between G protein-coupled receptors and EGFR for transactivation of EGFR. This is with a recent that signaling molecules, including the AT1 receptor and EGFR, by proteins is required for Ang II-induced transactivation of EGFR M. L. N. Y. K.K. Alexander R.W. J. Biol. Chem. Scholar). It be that phosphorylation of EGFR even interaction between the and EGFR is We that phosphorylation of EGFR is the of EGFR may be maintained through other mechanisms of are Although Ang II binding to the through an binding to AT1-Y319F through an mechanism the that AT1-Y319F failed to activate EGFR. The mechanism by which the mechanism for the of EGFR activation to activate in cells expressing AT1-Y319F is at be that EGFR may molecules leading to activation, Grb2, tyrosine 319 of the AT1 receptor is It be that has been shown that tyrosine kinases are able to Ang II-induced activation a protein kinase mechanism of activation is EMBO J. 1998; Scholar). tyrosine kinases may in a mechanism for the AT1 receptor to the of Our results that tyrosine 319 of the AT1 receptor plays an essential role in mediating Ang II-induced cell in cardiac Although we have shown in this that tyrosine 319 of the AT1 receptor plays a key role in Ang II-induced EGFR activation in cardiac this not the that activation of other molecules may also tyrosine 319. However, the that the Ang II-induced cell proliferation, is that the of the at tyrosine 319 is primarily through its Ang II-induced EGFR The signaling mechanisms of Ang II-induced have been primarily by using specific or molecules dominant 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). Our results suggest that the AT1 receptor mutant, which has a in the signaling be used to elucidate the of the signaling mechanisms activated by the AT1