Key points are not available for this paper at this time.
Several signal transduction events induced by angiotensin II (AngII) binding to the angiotensin II type 1 receptor resemble those evoked by platelet-derived growth factor (PDGF) binding to the PDGF-β receptor (PDGFβ-R). We report here, in agreement with previous data, that AngII and PDGF-B-chain homodimer (PDGF-BB) stimulate tyrosine phosphorylation of the PDGFβ-R. Both AngII and PDGF-BB stimulated the phosphorylation of PDGFβ-R via the binding of tyrosine-phosphorylated Shc to PDGFβ-R. Both PDGF-BB- and AngII-induced phosphorylation of the Shc·PDGFβ-R complex was inhibited by antioxidants such as N-acetylcysteine and Tiron, but not by calcium chelation. However, transactivation of PDGFβ-R by AngII (measured by PDGFβ-R tyrosine phosphorylation) differed significantly from PDGF-BB. Evidence to support different mechanisms of PDGFβ-R phosphorylation includes differences in the time course of PDGFβ-R phosphorylation, differing effects of inhibitors of the endogenous PDGFβ-R tyrosine kinase and Src family tyrosine kinases, differing results when the PDGFβ-R was directly immunoprecipitated (PDGFβ-R-antibody) versuscoimmunoprecipitated (Shc-antibody), and cell fractionation studies that suggested that the Shc·PDGFβ-R complexes phosphorylated by AngII and PDGF-BB were located in separate subcellular compartments. These studies are the first to suggest that transactivation of tyrosine kinase receptors by G protein-coupled receptors involves a unique pathway that regulates a population of tyrosine kinase receptors different from the endogenous tyrosine kinase ligand. Several signal transduction events induced by angiotensin II (AngII) binding to the angiotensin II type 1 receptor resemble those evoked by platelet-derived growth factor (PDGF) binding to the PDGF-β receptor (PDGFβ-R). We report here, in agreement with previous data, that AngII and PDGF-B-chain homodimer (PDGF-BB) stimulate tyrosine phosphorylation of the PDGFβ-R. Both AngII and PDGF-BB stimulated the phosphorylation of PDGFβ-R via the binding of tyrosine-phosphorylated Shc to PDGFβ-R. Both PDGF-BB- and AngII-induced phosphorylation of the Shc·PDGFβ-R complex was inhibited by antioxidants such as N-acetylcysteine and Tiron, but not by calcium chelation. However, transactivation of PDGFβ-R by AngII (measured by PDGFβ-R tyrosine phosphorylation) differed significantly from PDGF-BB. Evidence to support different mechanisms of PDGFβ-R phosphorylation includes differences in the time course of PDGFβ-R phosphorylation, differing effects of inhibitors of the endogenous PDGFβ-R tyrosine kinase and Src family tyrosine kinases, differing results when the PDGFβ-R was directly immunoprecipitated (PDGFβ-R-antibody) versuscoimmunoprecipitated (Shc-antibody), and cell fractionation studies that suggested that the Shc·PDGFβ-R complexes phosphorylated by AngII and PDGF-BB were located in separate subcellular compartments. These studies are the first to suggest that transactivation of tyrosine kinase receptors by G protein-coupled receptors involves a unique pathway that regulates a population of tyrosine kinase receptors different from the endogenous tyrosine kinase ligand. angiotensin II angiotensin type 1 receptor G-protein coupled receptor vascular smooth muscle cells extracellular signal-regulated kinases janus kinase epidermal growth factor epidermal growth factor receptor platelet-derived growth factor B-chain homodimer platelet-derived growth factor β receptor tumor necrosis factor 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl reactive oxygen species Angiotensin II (AngII),1an octapeptide pressor hormone, activates cellular events that may contribute to the pathogenesis of cardiovascular disease (1.Griendling K.K. Ushio F.-M. Lassegue B. Alexander R.W. Hypertension. 1997; 29: 366-373Crossref PubMed Google Scholar, 2.Bernstein K.E. Berk B.C. Am. J. Kidney Dis. 1993; 22: 745-754Abstract Full Text PDF PubMed Scopus (88) Google Scholar). The physiological actions of AngII are mediated largely via the AngII type 1 receptor (AT1R) (3.Oliverio M.I. Best C.F. Kim H.S. Arendshorst W.J. Smithies O. Coffman T.M. Am. J. Physiol. 1997; 272: F515-F520PubMed Google Scholar), which is a G protein-coupled receptor (GPCR). GPCRs share a common basic structure of seven transmembrane helices connected by alternating cytoplasmic and extracellular loops (4.Baldwin J.M. Curr. Opin. Cell Biol. 1994; 6: 180-190Crossref PubMed Scopus (340) Google Scholar). AngII-mediated growth effects in target cells such as vascular smooth muscle cells (VSMC) and cardiac myocytes require the rapid activation of several mitogen-activated protein kinases including the extracellular signal-regulated kinases (ERK1/2) (5.Weber H. Taylor D.S. Molloy C.J. J. Clin. Invest. 1994; 93: 788-798Crossref PubMed Scopus (145) Google Scholar). The signal transduction pathway that leads to ERK1/2 activation upon AngII binding to the AT1R is still incompletely characterized. There is evidence that binding of AngII to the AT1R stimulates tyrosine phosphorylation of several proteins including Shc and GRB-2 prior to activation of members of the Ras family (6.Sadoshima J. Izumo S. EMBO J. 1996; 15: 775-787Crossref PubMed Scopus (232) Google Scholar). Rapid tyrosine phosphorylation is likely mediated by several tyrosine kinases including Src family tyrosine kinases, JAK2 and PYK2 (7.Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (879) Google Scholar, 8.Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar, 10.Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Interestingly, certain aspects of the signal transduction pathways induced by AngII resemble those evoked by classic mitogenic growth factors (9.Berk B.C. Alexander R.W. Brock T.A. Gimbrone M.A. Webb Jr., R.C. Science. 1986; 232: 87-90Crossref PubMed Scopus (340) Google Scholar). In fact, recent studies show that GPCRs, including the AT1R, transactivate growth factor receptors, which possess intrinsic tyrosine kinase activity, including the EGF receptor (EGF-R) and PDGF receptor (PDGF-R) (10.Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 12.Murasawa 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: 1338-1348Crossref PubMed Scopus (180) Google Scholar). It has been suggested that the EGF-R and the PDGF-R mediate several of the cellular effects of AngII (8.Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar, 11.van Biesen T. Hawes B.E. Luttrell D.K. Krueger K.M. Touhara K. Porfiri E. Sakaue M. Luttrell L.M. Lefkowitz R.J. Nature. 1995; 376: 781-784Crossref PubMed Scopus (525) Google Scholar). In cardiac fibroblasts, Moriguchi et al. (13.Moriguchi Y. Matsubara H. Mori Y. Murasawa S. Masaki H. Maruyama K. Tsutsumi Y. Shibasaki Y. Tanaka Y. Circ. Res. 1999; 84: 1073-1084Crossref PubMed Scopus (109) Google Scholar) showed recently that ERK1/2 activation by AngII was mediated via EGF-R. Further elucidation of the mechanisms of transactivation of the EGF-R by AngII has revealed a Ca2+/calmodulin-dependent process that involves the endogenous EGF-R tyrosine kinase (12.Murasawa 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: 1338-1348Crossref PubMed Scopus (180) Google Scholar). Phosphorylation of the PDGFβ-R by AngII in VSMC has been described previously by Linseman (10.Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), but the mechanisms for PDGFβ-R phosphorylation remain poorly defined. In the present study, we compared the signaling events involved in AngII-induced PDGFβ-R transactivation with events involved in PDGF-B-chain homodimer (PDGF-BB)-mediated tyrosine phosphorylation of the PDGFβ-R. We show that AngII stimulates phosphorylation of the PDGFβ-R via the adaptor protein Shc more rapidly than PDGF itself and that PDGFβ-R phosphorylation by AngII is not dependent on calcium. Both Shc and PDGFβ-R phosphorylation induced by PDGF-BB and AngII were completely abolished by the antioxidants Tiron and N-acetylcysteine. However, phosphorylation of the PDGFβ-R by AngII and PDGF-BB occurred via different pathways as shown by different subcellular location and sensitivity to kinase inhibitors. Cell culture media and protein G-agarose were from Life Technologies, Inc. Polyclonal antibody against Shc, monoclonal anti-phosphotyrosine antibody (4G10), and polyclonal antibody against PDGFβ-R were from Upstate Biotechnology (Lake Placid, NY). Polyclonal anti-phospho-specific ERK1/2 antibody was from New England Biolabs. Recombinant (human) TNF and recombinant (human) PDGF-BB were from Sigma. BAPTA-AM, AG1296 (PDGF-R tyrosine kinase inhibitor), and PP-1 were from Calbiochem. Rat aortic VSMC were isolated from the thoracic aorta of 200–250-g male Harlan Sprague-Dawley rats and maintained in Dulbecco's modified Eagle's medium supplemented with 10% serum as described (14.Ishida M. Ishida T. Thomas S. Berk B.C. Circ. Res. 1998; 82: 7-12Crossref PubMed Scopus (150) Google Scholar). Passages 8–14 at 60–70% confluence were growth arrested by incubation in Dulbecco's modified Eagle's medium with 0.1% serum for 48 h before use. The methods for immunoprecipitation and immunoblot analysis were described previously (15.Schmitz U. Ishida T. Ishida M. Surapisitchat J. Hasham M.I. Pelech S. Berk B.C. Circ. Res. 1998; 82: 1272-1278Crossref PubMed Scopus (84) Google Scholar). In brief, growth-arrested VSMC were stimulated with AngII or PDGF-BB as indicated in each experiment. Cells were lysed in Triton/Nonidet P-40 lysis buffer (0.5% Triton, 0.5% Nonidet P-40, 10 mm Tris (pH 7.5), 2.5 mm KCl, 150 mm NaCl, 20 mm β-glycerol phosphate, 50 mm NaF, 1 mm orthovanadate, 10 μg/ml leupeptin, 1 mm dithiothreitol, 10 μg/ml soybean trypsin inhibitor, and 200 mm benzamidine), scraped off the dish, and centrifuged. Lysates containing equal amounts of protein were precleared and incubated with antibodies overnight at 4 °C. Antibody complexes were collected by incubation with protein G-agarose for 3 h at 4 °C. Precipitates were washed four times with the Triton/Nonidet P-40 lysis buffer and then resuspended in SDS sample buffer. Samples were separated by SDS-polyacrylamide gel electrophoresis (8–12%) and transferred to nitrocellulose membranes. After incubation in blocking solution (1% bovine serum albumin, 10 mm Tris (pH 7.5), 100 mm NaCl, 0.1% Tween 20), membranes were incubated with primary antibodies. After washing (Tris-buffered saline, 0.03% Tween 20), the blots were incubated with the appropriate secondary antibodies. The membranes were washed and proteins were detected by the ECL system (Amersham Pharmacia Biotech). To compare phosphorylation of the PDGβ-R by AngII and PDGF-BB, the PDGFβ-R was immunoprecipitated from cell lysates treated with AngII (100 nm) or PDGF-BB (30 ng/ml) for 2 min. Immunoblotting with an antibody against tyrosine-phosphorylated proteins showed phosphorylation of a ∼180-kDa band (Fig.1 A, upper panel) which was identified as the PDGFβ-R by stripping and reprobing with PDGFβ-R antibody (Fig. 1 A, lower panel). Phosphorylation of the PDGFβ-R by AngII at 2 min was ∼50% of phosphorylation by PDGF-BB itself. The adaptor protein Shc exists as three isoforms, 66 kDa, 52 kDa, and 46 kDa, and is a likely candidate to mediate cross-talk between the PDGFβ-R and the AT1R by assembling a signal transduction complex at the PDGFβ-R (7.Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (879) Google Scholar, 10.Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Therefore, AngII-induced PDGFβ-R phosphorylation may be studied by coimmunoprecipitation of Shc and the PDGFβ-R. AngII and PDGF-BB both stimulated tyrosine phosphorylation of only the 66-kDa isoform of Shc (Fig. 1 B, upper panel), whereas the two smaller Shc isoforms were not involved in PDGFβ-R transactivation in our cell system. Furthermore, tyrosine-phosphorylated Shc was part of a signal transduction complex that included the PDGFβ-R as shown by coimmunoprecipitation of the PDGFβ-R with Shc (Fig. 1 B, lower panel). To determine whether the signaling events induced by AngII and PDGF-BB which cause PDGFβ-R phosphorylation were similar, the time course for tyrosine phosphorylation of the PDGFβ-R was studied. AngII caused a more rapid and transient phosphorylation of the PDGFβ-R than did PDGF-BB (Fig. AngII-induced phosphorylation of the PDGFβ-R at 1 min and to 10 when immunoprecipitated with antibody against the PDGFβ-R. in PDGFβ-R phosphorylation was when with antibody against Shc (Fig. that of the PDGFβ-R phosphorylated in to AngII was to Shc, and was not In PDGF-BB phosphorylated the PDGFβ-R at 2 and phosphorylation was to min (Fig. and not Phosphorylation of the 66-kDa isoform of Shc by AngII was more rapid than phosphorylation of the PDGFβ-R not AngII calcium T. Y. Res. 1998; PubMed Scopus Google Scholar, T. J. Am. J. Physiol. 1994; PubMed Google Scholar) and the transactivation of the EGF-R by AngII was to be (12.Murasawa 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: 1338-1348Crossref PubMed Scopus (180) Google Scholar), we the of calcium in PDGFβ-R with BAPTA-AM, a of did not phosphorylation of the PDGFβ-R by AngII or PDGF-BB when with an antibody against Shc (Fig. and or when immunoprecipitated with an antibody against PDGFβ-R (Fig. 3 A, upper and 3 Furthermore, tyrosine phosphorylation of the 66-kDa Shc isoform was not significantly inhibited (Fig. 3 of ERK1/2 by both AngII and PDGF-BB was not in VSMC (Fig. 3 panel). stimulated ERK1/2 activation to a in of calcium activation of the kinase J. M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), we that activation of ERK1/2 in cells is a of in kinase a for calcium VSMC were treated with completely inhibited ERK1/2 activation by that with signaling events (Fig. 3 A, lower panel). It is that AngII stimulates reactive oxygen species in and in K.K. Circ. Res. 1999; PubMed Scopus Google Scholar). Furthermore, previous studies shown that are for signal transduction K. T. Science. 1995; 270: PubMed Scopus Google Scholar). To determine the of in AngII-induced Shc·PDGFβ-R phosphorylation, VSMC were with the N-acetylcysteine and Both antioxidants completely abolished tyrosine phosphorylation of the Shc·PDGFβ-R complex (Fig. To the of the endogenous PDGFβ-R tyrosine kinase in phosphorylation of the Shc·PDGFβ-R complex by we a and of kinase M. M. A. A. 1997; PubMed Scopus Google Scholar). we studied the of AG1296 on phosphorylation of PDGFβ-R that was with Shc as by immunoprecipitation with to events M. M. A. A. 1997; PubMed Scopus Google Scholar), AG1296 completely inhibited tyrosine phosphorylation of PDGFβ-R and Shc induced by PDGF-BB In AG1296 did not AngII-induced phosphorylation of the Shc·PDGFβ-R complex (Fig. we studied the of AG1296 on PDGFβ-R phosphorylation by immunoprecipitation with antibody (Fig. AG1296 caused of the AngII-induced tyrosine phosphorylation of the PDGFβ-R. These results suggest that two different of the PDGFβ-R by whether the PDGFβ-R is to the 66-kDa Shc isoform or is not The endogenous PDGFβ-R tyrosine kinase is for phosphorylation of the PDGFβ-R but not for phosphorylation of the Shc·PDGFβ-R complex studies from our and showed that the Src tyrosine kinase family is for and signaling events in such as at T. Ishida M. J. M. Berk B.C. J. Clin. Invest. 1999; PubMed Scopus Google Scholar), J. A. J. H. U. A. A. Circ. Res. 1999; PubMed Scopus Google Scholar), and J. A. J. H. U. A. A. Circ. Res. 1999; PubMed Scopus Google Scholar). To determine the of Src in and phosphorylation of the Shc·PDGFβ-R we a that with Src family kinases and is a of J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). PP-1 did not AngII-induced phosphorylation of the PDGFβ-R upper panel), AngII phosphorylation of Shc (Fig. A, panel). In PP-1 completely inhibited PDGFβ-R phosphorylation and Shc phosphorylation (Fig. in agreement with of et al. J. A. J. H. U. A. A. Circ. Res. 1999; PubMed Scopus Google Scholar). PP-1 completely inhibited the tyrosine phosphorylation of PDGFβ-R not to Shc that was stimulated by AngII (Fig. These results support the that PDGF-BB and AngII the PDGFβ-R via two different pathways in by with To the kinases involved in AngII-mediated Shc·PDGFβ-R complex phosphorylation, we which is rapidly by AngII N. T. H. M. E. M. A. A. A. Nature. 1996; PubMed Scopus Google Scholar). a JAK2 B. B. J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, N. T. H. M. E. M. A. A. A. Nature. 1996; PubMed Scopus Google Scholar), did not phosphorylation of the PDGFβ-R induced by PDGF-BB on AngII-mediated PDGFβ-R phosphorylation by Shc coimmunoprecipitation (Fig. These that JAK2 is not for PDGFβ-R phosphorylation by for different pathways of Shc·PDGFβ-R complex phosphorylation by AngII and PDGF-BB be that the Shc·PDGFβ-R complex is to a different subcellular when stimulated with is by the of et al. E. C. G. B. Biol. 1996; 16: PubMed Scopus Google Scholar), showed that Shc proteins were to membranes and activation of tyrosine kinase To determine whether separate of Shc·PDGFβ-R complexes be we cell fractionation PDGFβ-R was present in the of lysates at for min from cells stimulated with both AngII and PDGF-BB (Fig. A, panel). In tyrosine-phosphorylated PDGFβ-R was present in the of lysates from cells at for 10 min (Fig. A, panel). However, when cells were stimulated with PDGF-BB, tyrosine-phosphorylated PDGFβ-R was still present in the (Fig. A, panel). To determine the location of Shc AngII and PDGF-BB we the in P-40 lysis buffer and immunoprecipitated Shc with an shown in B, the Shc·PDGFβ-R complex was only present in the of cells stimulated with In lysates only a of Shc was present in the These results suggest that the PDGFβ-R phosphorylated in to AngII as a Shc·PDGFβ-R was located in a different from the PDGFβ-R phosphorylated in to PDGF-BB. We the amounts of present in the Shc·PDGFβ-R complexes from and was in not we not that to the between AngII and PDGF-BB. The of the present are that AngII and PDGF-BB stimulate tyrosine phosphorylation of the PDGFβ-R via different pathways (Fig. transactivation of the PDGFβ-R and EGF-R by AngII has been previously (10.Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 12.Murasawa S. Mori Y. Nozawa Y. Gotoh N. Shibuya M. Masaki H. Maruyama K. Tsutsumi Y. Moriguchi Y. Shibazaki Y. Tanaka Y. Iwasaka T. 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Res. 1998; 82: 1338-1348Crossref PubMed Scopus (180) Google Scholar), the of the present is the of two different pathways that are separate as shown by the of two of the PDGFβ-R. population of PDGFβ-R exists in which Shc is to the receptor in the whereas in the population the PDGFβ-R is not to Evidence to support different mechanisms for PDGFβ-R phosphorylation to separate includes differences in the time course of PDGFβ-R phosphorylation (Fig. differing effects of inhibitors of the endogenous PDGFβ-R tyrosine kinase and Src family tyrosine kinases 4 and differing results when the PDGFβ-R was directly immunoprecipitated (PDGFβ-R-antibody) versuscoimmunoprecipitated (Shc-antibody), and cell fractionation studies that suggested that the PDGFβ-R phosphorylated by AngII and PDGF-BB is located in separate subcellular (Fig. These studies are the first to suggest that transactivation of tyrosine kinase receptors by GPCRs involves a unique pathway that regulates a population of tyrosine kinase receptors different from the endogenous tyrosine kinase (Fig. In the the different mechanisms for and PDGFβ-R tyrosine phosphorylation are on the shown in It has that mediate of the rapid of VSMC to K.K. 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C. G. B. Biol. 1996; 16: PubMed Scopus Google Scholar). Shc was in cells with a of the that the of Shc with in the cytoplasmic of the receptor is to Shc to the cell The for Shc by GPCRs, to be There is evidence that the different Shc proteins and are in different of the cell E. S. G. G. T. EMBO J. 1997; 16: PubMed Scopus Google Scholar). et al. S. J. Biol. 1998; PubMed Scopus Google Scholar) showed that in the 66-kDa isoform was to the the isoform to the and the isoform was the S. J. Biol. 1998; PubMed Scopus Google Scholar). It is that GPCRs and tyrosine kinase receptors to a Shc However, in the present study, both AngII and PDGF-BB stimulated phosphorylation of 66-kDa Shc with phosphorylation of the and In the different of receptor phosphorylation by a tyrosine kinase the of or an intrinsic receptor tyrosine kinase (PDGF-BB) may the subcellular location of of the EGF-R by AngII in cardiac has been shown to be in the of and growth (13.Moriguchi Y. Matsubara H. Mori Y. Murasawa S. 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Heeneman et al. (Mon,) studied this question.