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• W2083712812 abstract "The low density lipoprotein receptor-related protein (LRP) functions in the catabolism of numerous ligands including proteinases, proteinase inhibitor complexes, and lipoproteins. In the current study we provide evidence indicating an expanded role for LRP in modulating cellular signaling events. Our results show that platelet-derived growth factor (PDGF) BB induces a transient tyrosine phosphorylation of the LRP cytoplasmic domain in a process dependent on PDGF receptor activation and c-Src family kinase activity. Other growth factors, including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1, were unable to mediate tyrosine phosphorylation of LRP. The basis for this selectivity may result from the ability of LRP to bind PDGFBB, because surface plasmon resonance experiments demonstrated that only PDGF, and not basic fibroblast growth factor, epidermal growth factor, or insulin-like growth factor-1, bound to purified LRP immobilized on a sensor chip. The use of LRP mini-receptor mutants as well as in vitro phosphorylation studies demonstrated that the tyrosine located within the second NPXY motif found in the LRP cytoplasmic domain is the primary site of tyrosine phosphorylation by Src and Src family kinases. Co-immunoprecipitation experiments revealed that PDGF-mediated tyrosine phosphorylation of LRPs cytoplasmic domain results in increased association of the adaptor protein Shc with LRP and that Shc recognizes the second NPXY motif within LRPs cytoplasmic domain. In the accompanying paper, Boucher et al. (Boucher, P., Liu, P. V., Gotthardt, M., Hiesberger, T., Anderson, R. G. W., and Herz, J. (2002) J. Biol. Chem. 275, 15507–15513) reveal that LRP is found in caveolae along with the PDGF receptor. Together, these studies suggest that LRP functions as a co-receptor that modulates signal transduction pathways initiated by the PDGF receptor. The low density lipoprotein receptor-related protein (LRP) functions in the catabolism of numerous ligands including proteinases, proteinase inhibitor complexes, and lipoproteins. In the current study we provide evidence indicating an expanded role for LRP in modulating cellular signaling events. Our results show that platelet-derived growth factor (PDGF) BB induces a transient tyrosine phosphorylation of the LRP cytoplasmic domain in a process dependent on PDGF receptor activation and c-Src family kinase activity. Other growth factors, including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1, were unable to mediate tyrosine phosphorylation of LRP. The basis for this selectivity may result from the ability of LRP to bind PDGFBB, because surface plasmon resonance experiments demonstrated that only PDGF, and not basic fibroblast growth factor, epidermal growth factor, or insulin-like growth factor-1, bound to purified LRP immobilized on a sensor chip. The use of LRP mini-receptor mutants as well as in vitro phosphorylation studies demonstrated that the tyrosine located within the second NPXY motif found in the LRP cytoplasmic domain is the primary site of tyrosine phosphorylation by Src and Src family kinases. Co-immunoprecipitation experiments revealed that PDGF-mediated tyrosine phosphorylation of LRPs cytoplasmic domain results in increased association of the adaptor protein Shc with LRP and that Shc recognizes the second NPXY motif within LRPs cytoplasmic domain. In the accompanying paper, Boucher et al. (Boucher, P., Liu, P. V., Gotthardt, M., Hiesberger, T., Anderson, R. G. W., and Herz, J. (2002) J. Biol. Chem. 275, 15507–15513) reveal that LRP is found in caveolae along with the PDGF receptor. Together, these studies suggest that LRP functions as a co-receptor that modulates signal transduction pathways initiated by the PDGF receptor. low density lipoprotein (LDL) receptor-related protein apolipoprotein E platelet-derived growth factor PDGF receptor fibroblast growth factor basic FGF insulin-like growth factor-1 epidermal growth factor receptor-associated protein hemagglutinin Dulbecco's modified Eagle's medium horseradish peroxidase glutathione S-transferase wild type phosphotyrosine binding disabled The low density lipoprotein receptor-related protein (LRP)1 is a large endocytic receptor containing a 515-kDa heavy chain to which ligands bind and a non-covalently associated 85-kDa light chain containing a transmembrane and cytoplasmic domain (for review see Ref. 1Herz J. Strickland D.K. J. Clin. Invest. 2001; 108: 779-784Crossref PubMed Scopus (887) Google Scholar). LRP is one of 12 or more receptors that make up the LDL receptor superfamily and is essential for embryonic development in mice (2Herz J. Clouthier D.E. Hammer R.E. Cell. 1992; 71: 411-421Abstract Full Text PDF PubMed Scopus (508) Google Scholar). A remarkable feature of LRP is its ability to bind and mediate the internalization of a diverse array of ligands, including proteinases (3Kounnas M.Z. Henkin J. Argraves W.S. Strickland D.K. J. Biol. Chem. 1993; 268: 21862-21867Abstract Full Text PDF PubMed Google Scholar, 4Hahn-Dantona E. Ruiz J.F. Bornstein P. Strickland D.K. J. Biol. Chem. 2001; 276: 15498-15503Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), proteinase-inhibitor complexes (5Nykjær A. Petersen C.M. Moller B. Jensen P.H. Moestrup S.K. Holtet T.L. Etzerodt M. Thogersen H.C. Munch M. Andreasen P.A. Gliemann J. J. Biol. Chem. 1992; 267: 14543-14546Abstract Full Text PDF PubMed Google Scholar, 6Kounnas M.Z. Church F.C. Argraves W.S. Strickland D.K. J. Biol. Chem. 1996; 271: 6523-6529Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), and lipoproteins (7Rohlmann A. Gotthardt M. Hammer R.E. Herz J. J. Clin. Invest. 1998; 101: 689-695Crossref PubMed Scopus (400) Google Scholar). After binding to the LRP, the ligands are transported into endosomes where they uncouple in the reduced pH environment and are sorted to lysosomes for degradation. LRP recycles back to the cell surface where it is once again available to bind ligands. Recent studies indicate that in addition to their cargo transport function, certain LDL receptor family members also participate in signaling pathways. For example, the very low density lipoprotein receptor and apoE receptor 2 both participate in a signal transduction pathways mediated by reelin (8Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R.E. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar, 9Hiesberger T. Trommsdorff M. Howell B.W. Goffinet A. Mumby M.C. Cooper J.A. Herz J. Neuron. 1999; 24: 481-489Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar, 10D'Arcangelo G. Homayouni R. Keshvara L. Rice D.S. Sheldon M. Curran T. Neuron. 1999; 24: 471-479Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar). Reelin is secreted by Cajal-Retzius cell in the outermost layer of the cerebral cortex and controls the final position of neurons that migrate from the ventricular zone. Binding of reelin to either the very low density lipoprotein receptor or apoE receptor 2 induces tyrosine phosphorylation of disabled-1 (Dab1) (9Hiesberger T. Trommsdorff M. Howell B.W. Goffinet A. Mumby M.C. Cooper J.A. Herz J. Neuron. 1999; 24: 481-489Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar, 10D'Arcangelo G. Homayouni R. Keshvara L. Rice D.S. Sheldon M. Curran T. Neuron. 1999; 24: 471-479Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar), an adaptor protein that interacts with the cytoplasmic domains of LDL receptor family members (11Trommsdorff R. Borg J.P. Margolis B. Herz J. J. Biol. Chem. 1998; 273: 33556-33560Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar, 12Howell B.W. Lanier L.M. Frank R. Gertler F.B. Cooper J.A. Mol. Cell. Biol. 1999; 19: 5179-5188Crossref PubMed Scopus (335) Google Scholar) and functions in tyrosine kinase signaling pathways. In the case of LRP, accumulating evidence suggests a prominent but undefined role for this receptor in regulating cell physiology by facilitating signal transduction pathways. For example, LRP has been implicated as a component of the receptor complex for midkine (13Muramatsu H. Zou K. Sakaguchi N. Ikematsu S. Sakuma S. Muramatsu T. Biochem. Biophys. Res. Commun. 2000; 270: 936-941Crossref PubMed Scopus (132) Google Scholar), a heparin binding growth factor with migration-promoting and survival-promoting activities. Another LRP ligand, tissue type plasminogen activator, promotes late phase long term potentiation (14Huang Y.Y. Bach M.E. Lipp H.P. Zhuo M. Wolfer D.P. Hawkins R.D. Schoonjans L. Kandel E.R. Godfraind J.M. Mulligan R. Collen D. Carmeliet P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8699-8704Crossref PubMed Scopus (282) Google Scholar), and this activity appears to require its association with LRP (15Zhuo M. Li D.M. Holtzman Y. Osaka H. DeMaro J. Jacquin M. Bu G. J. Neurosci. 2000; 20: 542-549Crossref PubMed Google Scholar). Finally, the binding of activated α2M (α2M*) to LRP mediates calcium influx in neurons in a process that also involves N-methyl-d-aspartate receptors (16Bacskai B.J. Xia M.Q. Strickland D.K. Rebeck G.W. Hyman B.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11551-11556Crossref PubMed Scopus (172) Google Scholar). The exact role LRP plays in all of these processes is not known. Recently, Barnes et al. (17Barnes H. Larsen B. Tyers M. van Der G.P. J. Biol. Chem. 2001; 22: 19119-19125Abstract Full Text Full Text PDF Scopus (97) Google Scholar) demonstrated that LRP is tyrosine-phosphorylated in v-Src-transformed cells and provided evidence suggesting that phosphorylated LRP binds to Shc, an adaptor protein that is important in the activation of the Ras (18Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Li R. Daly W. Batzer A. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (827) Google Scholar) and c-Myc signaling pathways (19Gotoh N. Toyoda M. Shibuya M. Mol. Cell. Biol. 1997; 17: 1824-1831Crossref PubMed Scopus (140) Google Scholar). In the present investigation we demonstrate that platelet derived growth factor (PDGF) BB directly binds to LRP and promotes the transient tyrosine phosphorylation of the LRP cytoplasmic domain via activation of the PDGF receptor and c-Src. This phosphorylation occurs on a tyrosine residue located within the second NPXY motif found in the LRP cytoplasmic domain and generates a docking site for adaptor proteins such as Shc. In the accompanying paper Boucheret al. (20Boucher P. Liu P.V. Gotthardt M. Hiesberger T. Anderson R.G.W. Herz J. J. Biol. Chem. 2002; 275: 15507-15513Abstract Full Text Full Text PDF Scopus (178) Google Scholar) demonstrate that, like the PDGF receptor, LRP also localizes in caveolae and the LRP ligand apoE-enriched β very low density lipoprotein blocks PDGF-mediated tyrosine phosphorylation of LRP. Taken together, these findings suggest an integrative co-receptor function between the PDGF receptor and LRP, indicating that LRP and certain of its ligands may modulate signal transduction pathways mediated by the PDGF receptor. A rabbit polyclonal IgG prepared against purified human LRP (R2629) was affinity-purified over LRP-Sepharose as described (21Kounnas M.Z. Morris R.E. Thompson M.R. Fitzgerald D.J. Strickland D.K. Saelinger C.B. J. Biol. Chem. 1992; 267: 12420-12423Abstract Full Text PDF PubMed Google Scholar). Monoclonal antibody 5A6, which recognizes the LRP light chain (or β subunit), was prepared against human LRP and has been described (22Strickland D.K. Ashcom J.D. Williams S. Burgess W.H. Migliorini M. Argraves W.S. J. Biol. Chem. 1990; 265: 17401-17404Abstract Full Text PDF PubMed Google Scholar). Cells producing the anti-Myc IgG 9E10 were obtained from the American Type Culture Collection (Manassas, VA), and the IgG was purified by chromatography on protein G-Sepharose. Anti-PDGF receptor β rabbit polyclonal IgG was purchased from Santa Cruz Biotechnology. The phosphotyrosine-specific monoclonal antibody 4G10 (23Cohen B. Yoakim M. Piwnica-Worms H. Roberts T.M. Schaffhausen B.S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4458-4462Crossref PubMed Scopus (65) Google Scholar) conjugated to horse-radish peroxidase and anti-Src rabbit polyclonal IgG were purchased from Upstate Biotechnology. Anti-Shc rabbit polyclonal IgG was purchased from Transduction Laboratories, whereas phospho-specific and total extracellular signal-regulated kinase polyclonal antibodies were obtained from New England Biolabs. Basic FGF, PDGFBB, PDGFAA, IGF-1, and EGF were purchased from R&D Systems. In all experiments, PDGFBB was utilized. LRP was isolated from human placenta as described by Ashcom et al. (24Ashcom J.D. Tiller S.E. Dickerson K. Cravens J.L. Argraves W.S. Strickland D.K. J. Cell Biol. 1990; 110: 1041-1048Crossref PubMed Scopus (205) Google Scholar) and labeled with [125I]iodine to a specific activity ranging from 2 to 10 μCi/μg protein using iodogen (Pierce). Human receptor-associated protein (RAP) was expressed in bacteria as fusion proteins with glutathione S-transferase and was cleaved and purified as described previously (25Williams S.E. Ashcom J.D. Argraves W.S. Strickland D.K. J. Biol. Chem. 1992; 267: 9035-9040Abstract Full Text PDF PubMed Google Scholar). The cytoplasmic domain of LRP was expressed as a fusion protein with glutathioneS-transferase in Escherichia coli using pGEX-2T expression vector (Promega). Construction of this expression vector was accomplished by preparing a cDNA fragment encoding amino acid residues 4426–4525 of human LRP (numbering is based on the mature protein, as defined in Herz et al. (26Herz J. Hamann U. Rogne S. Myklebost O. Gausepohl H. Stanley K.K. EMBO J. 1988; 7: 4119-4127Crossref PubMed Scopus (739) Google Scholar)) by polymerase chain reaction using 21-base synthetic oligonucleotide primers and an LRP cDNA (27Ulery P.G. Beers J. Mikhailenko I. Tanzi R.E. Rebeck G.W. Hyman B.T. Strickland D.K. J. Biol. Chem. 2000; 275: 7410-7415Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) as a template. The fusion protein was expressed and purified as described (25Williams S.E. Ashcom J.D. Argraves W.S. Strickland D.K. J. Biol. Chem. 1992; 267: 9035-9040Abstract Full Text PDF PubMed Google Scholar). Substitutions of asparagine and tyrosine to alanines in the two NPXY motifs of the cytoplasmic domain of the GST cytoplasmic domain were performed using Transformer site-directed mutagenesis kit (CLONTECH) and confirmed by sequencing. The cDNA of human LRP (27Ulery P.G. Beers J. Mikhailenko I. Tanzi R.E. Rebeck G.W. Hyman B.T. Strickland D.K. J. Biol. Chem. 2000; 275: 7410-7415Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) was also used as a template to generate expression vectors for the LRP β essentially as described (28Mikhailenko I. Battey F.D. Migliorini M. Ruiz J.F. Argraves K. Moayeri M. Strickland D.K. J. Biol. Chem. 2001; 276: 39484-39491Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Briefly, the fragment of cDNA that encodes amino acids 3844–4525 of LRP (GenBankTM access number X13916) was generated by PCR amplification and subcloned into pSecTag expression vector (Invitrogen) modified to produce a protein with two copies of Myc epitope at its amino terminus. The mini-receptor contains a portion of the LRP extracellular domain (including membrane proximal YWTD β-propeller and EGF-like repeats), transmembrane domain, and cytoplasmic tail. Substitutions of asparagine and tyrosine to alanines in the two NPXY motifs of the cytoplasmic domain of the mini-receptor were performed using the Transformer site-directed mutagenesis kit (CLONTECH) and confirmed by sequencing. All PCR products were sequenced before using to confirm that no errors were introduced by the PCR. Expression constructs encoding wild-type c-Src and kinase-inactive c-Src (K279R) in pUSEamp(−) were purchased from Upstate Biotechnology. A plasmid containing HA-tagged Shc was a generous gift from Dr. K. S. Ravichandran (University of Virginia, Charlotte, VA). Microtiter wells were coated with PDGFBB (2 μg/ml in Tris-buffered saline (TBS), pH 8.0, 50 μl/well) overnight and then blocked with 300 μl of 3% bovine serum albumin in TBS. 100 μl of 125I-labeled LRP (200 nm) was then added to the wells in the absence or presence of RAP (20 μm) and incubated overnight at 4 °C. After incubation, the microtiter wells were washed and counted. Binding of PDGFBB, bFGF, EGF, IGF-1 to purified LRP was measured using a BIA 3000 optical biosensor (Biacore AB, Uppsala, Sweden). For these studies, the BIAcore sensor chip (type CM5; Biacore AB) was activated with a 1:1 mixture of 0.2mN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide and 0.05 mN-hydroxysuccinimide in water as described by the manufacturer. Purified human LRP was immobilized at the level of 3000 response units in a working solution of 10 μg/ml in 10 mm sodium acetate, pH 4.0, through the BIAcore flow cell at a rate of 5 μl/min. The remaining binding sites were blocked by 1 m ethanolamine, pH 8.5, whereas unbound protein was washed out with 0.5% SDS. An additional flow cell, similarly activated and blocked without immobilization of protein, served as a negative control. A flow cell with immobilized ovalbumin at the level of 500 response units was used as a control for nonspecific protein binding. All binding reactions were performed in 10 mm HEPES, 0.15 m NaCl, 0.005% Tween 20, pH 7.4 (HBS-P buffer) (Biacore AB), containing 0.005% Tween 20. Binding of PDGFBB and selected growth factors to LRP was measured at 25 °C at a flow rate of 30 μl/min for 4 min, followed by 4 min of dissociation. The bulk shift due to changes in refractive index measured on blank surfaces was subtracted from the binding signal at each condition to correct for nonspecific signals. Chip surfaces were regenerated with subsequent 1-min pulses of 10 mm sodium acetate, pH 4.0, containing 1 m NaCl and 10 mm NaOH containing 1m NaCl followed by 2 min of washing with running buffer to remove this high salt solution. All injections were performed using Application Wizard in the automated method. Binding of PDGFBB was measured using 2-fold dilutions in HBS-P buffer over a range of concentrations (20–0.6 nm). Other growth factors as bFGF, EGF, and IGF-1 were injected at concentrations of 50 nm. All collected data were analyzed with BIA evaluation 3.0 software (Biacore) using global analysis to fit 1:1 Langmuir binding with mass transfer limitation and heterogeneous ligand models. WI-38 fibroblasts were cultured in 150-mm plates in DMEM containing 10% serum until they reached 60–70% confluency. Cell layers were then washed three times with serum-free medium. After washing, the media was replaced with DMEM containing either 0.1% fetal bovine serum or 1% Nutridoma® NS, and the cells were incubated with this media for an additional 18 h. For PDGF treatment, PDGFBB was added to the cells in DMEM containing either 0.1% fetal bovine serum or 1% Nutridoma® NS. After stimulation, cell layers were washed 2 times with cold Dulbecco’s phosphate-buffered saline containing 1 mm orthovanadate, and the lysate was prepared in lysis buffer (50 mm Tris, 150 mm NaCl, 1% Nonidet P-40, and a protease and phosphatase inhibitor mixture (Calbiochem)). After preclearing with mouse (or rabbit), IgG (10 μg/ml) lysates were immunoprecipitated with either monoclonal 5A6-protein G-Sepharose or R2629-protein G-Sepharose. Immunoprecipitates were washed 3 times with lysis buffer and then boiled with nonreducing SDS-PAGE sample buffer for 10 min. Samples were separated by electrophoresis on 4–20% or 8% SDS-PAGE precast gels (Invitrogen) and transferred to nitrocellulose membranes for immunoblot analysis. Membranes were blocked with 2% bovine serum albumin in Dulbecco’s phosphate-buffered saline for 1 h and incubated with anti-phosphotyrosine monoclonal IgG 4G10-horseradish peroxidase (HRP) conjugate (Calbiochem) (1:3000 dilution) in 2% bovine serum albumin in Dulbecco’s phosphate-buffered saline with 0.1% Tween 20 for 1 h and washed 5 × 4 in Dulbecco’s phosphate-buffered saline with 0.1% Tween 20. Membranes were developed with chemiluminescent reagent (Pierce), and bands were visualized using Biomax MR film (Eastman Kodak Co.). For visualizing immunoprecipitated LRP, the membranes were first stripped with Re-blot Western blot recycling kit (Chemicon International), and then the membranes were probed with iodinated 5A6 (2 μg/ml) overnight, washed, and exposed to BiomaxMR film. Extracellular signal-regulated kinase activation was measured by immunoblotting cell lysates (25 μg/lane) with phospho-specific and total extracellular signal-regulated kinase polyclonal antibodies (New England Biolabs) according to manufacturer’s instructions. WI-38 cells were grown in 150-mm plates in DMEM containing 10% serum to 60–70% confluency. Cell layers were then washed 3 times with serum-free medium and then incubated in 1% Nutridoma containing DMEM overnight. After serum deprivation, cells were preincubated for 15 min with inhibitors PP2 (300 nm), PP3 (300 nm), and AG1296 (900 nm) followed by incubation with 30 ng/ml PDGFBB for 12–15 min. PP2, PP3, and AG1296 were obtained from Calbiochem. Cells were washed, lysed, and processed as mentioned above. COS-1 cells were cultured to 30% confluency and then transfected with 10 μg of HA-Shc containing plasmid using FuGENE 6 transfection reagent (Roche Molecular Biochemicals). 24 h after transfection, the cells were cultured for 18 h in DMEM medium containing 0.1% fetal bovine serum. The cells were then treated with PDGFBB (40 ng/ml), whereas control cells received no treatment. After 15 min, cell extracts were prepared and subjected to immunoprecipitation with anti-LRP polyclonal R2629, and immunoprecipitated proteins were subjected to immunoblot analysis with anti-phosphotyrosine IgG 4G10 and anti-HA IgG. Src kinase assay was carried out using purified active human recombinant Src kinase (Calbiochem) according to the manufacturer’s instructions. Briefly 10 μg of purified protein (GST-wild-type (WT), GST-(NPTY → APTA), GST-(NPVY → APVA), GST) was incubated with 10 units of purified Src and 10 μCi of [γ-32P]ATP for 10 min at 30 °C. The reactions were terminated by the addition of sample buffer containing β−mercaptoethanol. Phospho-labeled proteins were separated on 4–12% SDS-PAGE precast gel and blotted onto nitrocellulose membranes. The nitrocellulose membrane was stained with Ponseau S and then exposed to BiomaxMS film. WT LRP-β or each of NPTY → APTA, NPVY → APVA mutant LRP expression vectors were transiently transfected into COS-1 cells with either pUSE empty vector (mock) or one of the c-Src expression plasmids (c-Src(WT), c-Src K297R (kinase inactive)). After transfection, cell extracts were prepared and subjected to immunoprecipitation with anti-LRP monoclonal 5A6. The immunoprecipitated proteins were tested for tyrosine phosphorylation by immunoblotting with anti-phosphotyrosyl antibodies. Whole cell extracts (2%) were analyzed by immunoblotting for LRP-β expression using anti-Myc IgG and for c-Src expression using anti-Src IgG. When added to fibroblasts, PDGF binds to PDGF receptors and induces rapid dimerization and tyrosine phosphorylation of these receptors (29Claesson-Welsh L. J. Biol. Chem. 1994; 269: 32023-32026Abstract Full Text PDF PubMed Google Scholar). These events are followed by internalization and degradation of the receptor-ligand complex (30Sorkin A. Westermark B. Heldin C.H. Claesson-Welsh L. J. Cell Biol. 1991; 112: 469-478Crossref PubMed Scopus (127) Google Scholar). To determine whether PDGF induces phosphorylation of the LRP cytoplasmic domain, WI-38 fibroblasts were incubated with PDGF for varying time periods. Analysis of cell extracts for the presence of phosphotyrosine using the phosphotyrosine-specific monoclonal antibody 4G10 (23Cohen B. Yoakim M. Piwnica-Worms H. Roberts T.M. Schaffhausen B.S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4458-4462Crossref PubMed Scopus (65) Google Scholar) and for the PDGF receptor antigen identified the rapid tyrosine phosphorylation of a prominent band with a mobility identical to the PDGF receptor (Fig.1A). The phosphoprotein disappeared with time, as expected for the PDGF receptor. The same cell extracts were subjected to immunoprecipitation with anti-LRP IgG and probed for tyrosine phosphorylation. The results (Fig.1B) demonstrated that the LRP β chain becomes phosphorylated on tyrosine residues in response to PDGF treatment. Maximal LRP phosphorylation occurred 10 min after the addition of PDGF, and interestingly, the phosphorylation was transient and disappeared with time. These results indicate that binding of PDGF to cells results in a transient phosphorylation of the tyrosine residue(s) within the LRP cytoplasmic domain. Similar results were obtained in rat smooth muscle cells (data not shown). To determine whether other growth factors are also able to mediate phosphorylation of LRP, serum-starved fibroblasts were incubated with various growth factors before subjecting cell extracts to immunoprecipitation with anti-LRP monoclonal antibodies. Immunoprecipitates were then probed with anti-phosphotyrosine. The results demonstrate that only PDGF-treated fibroblasts contain tyrosine-phosphorylated LRP (Fig. 2,upper panel, lane 3). Other growth factors including bFGF, IGF-1, and EGF did not stimulate the tyrosine phosphorylation of LRP. When the membranes were stripped and reprobed with 125I-labeled anti-LRP IgG we confirmed that LRP was immunoprecipitated from the cell extracts (Fig. 2, middle panel). A control experiment confirmed that all growth factors were active because they were able to induce the phosphorylation of extracellular signal-regulated kinase (Fig. 2, lower panel). These results reveal selectivity for PDGF in mediating the tyrosine phosphorylation of the LRP cytoplasmic domain. To gain insight into possible mechanisms by which PDGF promotes LRP tyrosine phosphorylation, studies were initiated to determine whether LRP can directly bind PDGF. To accomplish this, the binding of PDGF and other growth factors to purified LRP immobilized on a BIACore sensor chip was measured. The sensorgrams, shown in Fig. 3A, demonstrate that PDGF, but not bFGF, EGF, and IGF, bound LRP immobilized on a sensor chip. The binding of PDGF but not other growth factors by LRP might offer an explanation for the selectivity in LRP phosphorylation observed in Fig. 2. The affinity of PDGF binding to LRP was estimated by injecting varying concentrations of PDGF on the LRP-immobilized chip (Fig. 3B), and the results demonstrate a concentration-dependent binding of PDGF to the sensor chip. The data did not fit a single site model but were adequately described by a model in which LRP contains two binding sites for PDGF. The binding is characterized by KD values of 12 and 17 nm, with one site displaying rapid association and dissociation rates, whereas the second site displayed slower association and dissociation rates. The binding of all known ligands to LRP is prevented by the 39-kDa RAP (25Williams S.E. Ashcom J.D. Argraves W.S. Strickland D.K. J. Biol. Chem. 1992; 267: 9035-9040Abstract Full Text PDF PubMed Google Scholar, 31Herz J. Goldstein J.L. Ho D.K. Strickland Y.K. Brown M.S. J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar). To determine whether the binding of PDGF to LRP is inhibited by RAP, an enzyme-linked immunosorbent assay was performed in which the binding of 125I-labeled purified LRP to microtiter wells coated with PDGF was examined. The results (Fig.4A) demonstrate that RAP partially inhibited the binding of LRP to immobilized PDGF and did not completely reduce the binding to background levels. At this time, we speculate that LRP contains two PDGF binding sites, with only one site sensitive to RAP inhibition. However, to prove this will require additional studies. We next measured the effect of RAP on PDGF-mediated phosphorylation of LRP. WI-38 fibroblasts were incubated with PDGF in the presence and absence of RAP, and the degree of tyrosine phosphorylation of the LRP cytoplasmic domain was measured. The results (Fig. 4B, lane 3) demonstrate that RAP slightly reduces but does not prevent the phosphorylation of LRP mediated by PDGF, consistent with the data presented in Fig. 4A. The ability of PDGF to bind to LRP raises the possibility that phosphorylation of LRP mediated by PDGF could result either from direct association of this growth factor with LRP or, alternatively, via an integrative interaction between the PDGF receptor and LRP. To further characterize the mechanism by which PDGF initiates tyrosine phosphorylation of LRP, several inhibitors were employed. The PDGF receptor is known to activate Src family kinases, and thus, we used the cell-permeable Src family kinase inhibitor PP2 to determine whether Src family members are involved in tyrosine phosphorylation of LRP. PP2 competes with ATP for binding to Src family kinases (32Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1784) Google Scholar), thereby inhibiting enzymatic activity. Fig. 5 shows that 300 nm PP2 inhibited LRP phosphorylation (Fig. 5, lane 3). In contrast, identical amounts of the structurally related PP3 had no effect in this assay (Fig. 5, lane 4). PP3 is an appropriate negative control since it does not alter Src family kinase activity. Together, these results suggest that Src family kinase members mediate the phosphorylation of the LRP cytoplasmic domain. To verify a role for the PDGF receptor in this process, we used the PDGF receptor-specific inhibitor, AG1296 (" @default.
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• W2083712812 date "2002-05-01" @default.
• W2083712812 modified "2023-10-10" @default.
• W2083712812 title "Platelet-derived Growth Factor (PDGF)-induced Tyrosine Phosphorylation of the Low Density Lipoprotein Receptor-related Protein (LRP)" @default.
• W2083712812 cites W1484985322 @default.
• W2083712812 cites W1493954171 @default.
• W2083712812 cites W152075184 @default.
• W2083712812 cites W1528782632 @default.
• W2083712812 cites W1539895365 @default.
• W2083712812 cites W1548272930 @default.
• W2083712812 cites W1554558913 @default.
• W2083712812 cites W1555380614 @default.
• W2083712812 cites W1556453331 @default.
• W2083712812 cites W1583346750 @default.
• W2083712812 cites W1588573376 @default.
• W2083712812 cites W1600508667 @default.
• W2083712812 cites W1607784649 @default.
• W2083712812 cites W1619700982 @default.
• W2083712812 cites W1673016819 @default.
• W2083712812 cites W1923431801 @default.
• W2083712812 cites W1966161684 @default.
• W2083712812 cites W1983376590 @default.
• W2083712812 cites W1983795337 @default.
• W2083712812 cites W1985413713 @default.
• W2083712812 cites W1986758681 @default.
• W2083712812 cites W1992894005 @default.
• W2083712812 cites W1997038287 @default.
• W2083712812 cites W2000269495 @default.
• W2083712812 cites W2019291050 @default.
• W2083712812 cites W2023395361 @default.
• W2083712812 cites W2029634948 @default.
• W2083712812 cites W2032359283 @default.
• W2083712812 cites W2033822739 @default.
• W2083712812 cites W2035026318 @default.
• W2083712812 cites W2039049403 @default.
• W2083712812 cites W2058071886 @default.
• W2083712812 cites W2059657578 @default.
• W2083712812 cites W2060043725 @default.
• W2083712812 cites W2063262154 @default.
• W2083712812 cites W2065429233 @default.
• W2083712812 cites W2067089828 @default.
• W2083712812 cites W2079396188 @default.
• W2083712812 cites W2090895449 @default.
• W2083712812 cites W2094247513 @default.
• W2083712812 cites W2102910674 @default.
• W2083712812 cites W2113438288 @default.
• W2083712812 cites W2125856024 @default.
• W2083712812 cites W2132246987 @default.
• W2083712812 cites W2165700649 @default.
• W2083712812 cites W3102875255 @default.
• W2083712812 cites W339018046 @default.
• W2083712812 cites W4248025115 @default.
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