RESUMO
Hepatocyte growth factor (HGF) is a potent epithelial mitogen whose actions are mediated through its receptor, the proto-oncogene c-Met. Two truncated variants of HGF known as NK1 and NK2 have been reported to be competitive inhibitors of HGF binding to c-Met, and to function as HGF antagonists (Lokker, N.A., and P.J. Godowski. 1993. J. Biol. Chem. 268: 17145-17150; Chan, A.M., J.S. Rubin, D.P. Bottaro, D.W. Hirschfield, M. Chedid, and S.A. Aaronson. 1991. Science (Wash. DC). 254:1382-1387). We show here, however, that NK1 acts as a partial agonist in mink lung cells. Interestingly, NK1, which is an HGF antagonist in hepatocytes in normal conditions, was converted to a partial agonist by adding heparin to the culture medium. The interaction of NK1 and heparin was further studied in BaF3 cells, which express little or no cell surface heparan sulfate proteoglycans. In BaF3 cells transfected with a plasmid encoding human c-Met, heparin and NK1 synergized to stimulate DNA synthesis and cell proliferation. There was no effect of heparin on the IL-3 sensitivity of BaF3-hMet cells, and no effect of NK1 plus heparin in control BaF3 cells, indicating that the response was specific and mediated through c-Met. The naturally occurring HGF splice variant NK2 also stimulated DNA synthesis in mink lung cells and exerted a heparin-dependent effect on BaF3-hMet cells, but not on BaF3-neo cells. The activating effect of heparin was mimicked by a variety of sulfated glycosaminoglycans. Mechanistic studies revealed that heparin increased the binding of NK1 to BaF3-hMet cells, stabilized NK1, and induced dimerization of NK1. Based on these studies, we propose that the normal agonist activity of NK1 and NK2 in mink lung cells is due to an activating interaction with an endogenous glycosaminoglycan. Consistent with that model, a large portion of the NK1 binding to mink lung cells could be blocked by heparin. Moreover, a preparation of glycosaminoglycans from the surface of mink lung cells induced dimerization of NK1. These data show that the activity of NK1 and NK2 can be modulated by heparin and other related glycosaminoglycans to induce proliferation in cells expressing c-Met.
Assuntos
Heparina/farmacologia , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fígado/citologia , Animais , Divisão Celular/efeitos dos fármacos , DNA/biossíntese , Feminino , Glicosaminoglicanos/farmacologia , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Pulmão/citologia , Vison , Ligação Proteica/fisiologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-met , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/fisiologia , Receptores da Neurocinina-1/agonistas , Receptores da Neurocinina-1/metabolismo , Receptores da Neurocinina-2/agonistas , Receptores da Neurocinina-2/metabolismo , Sensibilidade e EspecificidadeRESUMO
Transforming growth factor-beta (TGF-beta) affects cellular proliferation, differentiation, and interaction with the extracellular matrix primarily through interaction with the type I and type II TGF-beta receptors. The type II receptors for TGF-beta and activin contain putative serine-threonine kinase domains. A murine serine-threonine kinase receptor, Tsk 7L, was cloned that shared a conserved extracellular domain with the type II TGF-beta receptor. Overexpression of Tsk 7L alone did not increase cell surface binding of TGF-beta, but coexpression with the type II TGF-beta receptor caused TGF-beta to bind to Tsk 7L, which had the size of the type I TGF-beta receptor. Overexpression of Tsk 7L inhibited binding of TGF-beta to the type II receptor in a dominant negative fashion. Combinatorial interactions and stoichiometric ratios between the type I and II receptors may therefore determine the extent of TGF-beta binding and the resulting biological activities.
Assuntos
Receptores de Superfície Celular/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases , Codorniz , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Fatores de Crescimento Transformadores beta , TransfecçãoRESUMO
Elevated plasma levels of lipoprotein(a), Lp(a), represent a major, inherited risk factor for coronary heart disease, although the mechanism of its action remains unknown. Lp(a) is distinguished from the related LDL particle by the addition of apolipoprotein(a), apo(a). The presence of this large glycoprotein is likely to affect the binding of the particle to the LDL receptor and/or other receptors which may contribute to the atherogenic potential of Lp(a). Here we demonstrate the binding to macrophages of Lp(a) and pure recombinant apo(a) protein, via a specific, high-affinity receptor. This binding could lead to foam cell formation and the localization of Lp(a) to atherosclerotic plaques.
Assuntos
Apolipoproteínas/metabolismo , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Endocitose , Citometria de Fluxo , Humanos , Técnicas In Vitro , Lipoproteína(a) , Lipoproteínas LDL/metabolismo , Lisossomos/metabolismo , Camundongos , Proteínas Recombinantes/metabolismoRESUMO
Bone healing is regulated in part by the local production of TGF-beta 1 and other growth factors produced by cells at the site of injury. The single application of recombinant human TGF-beta 1 (rhTGF-beta 1) to calvarial defects in rabbits induces an accelerated recruitment and proliferation of osteoblasts within 3 days. This ultimately results in the formation of new bone and the complete closure of the defect within 28 days. The persistence and localization of [125I]rhTGF-beta 1 within an osseous defect was investigated after applying a single dose of [125I]rhTGF-beta 1 formulated in a 3% methylcellulose vehicle. Normal bone encompassing the defect site, the periosteum, and the gel film covering the dura were harvested at 0, 4, 8, and 24 h and 3, 7, and 16 days after [125I]rhTGF-beta 1 application. The defect site-associated radioactivity was quantitated, visualized by autoradiography, and characterized by TCA precipitation and SDS-PAGE. Radioactivity was observed in autoradiographs of gross specimens, histologic sections of the bone matrix, and periosteal tissue surrounding the defect. There was a time-dependent decrease in TCA-precipitable radioactivity; however, radioactivity was still associated with the bone matrix 16 days after application of [125I]rhTGF-beta 1. SDS-PAGE and autoradiography of the radioactivity in homogenized bone and periosteal samples revealed a 25 kD band, suggesting that the radioactivity remaining at the defect site represented intact [125I]rhTGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Consolidação da Fratura/efeitos dos fármacos , Crânio/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Radioisótopos do Iodo/metabolismo , Masculino , Coelhos , Crânio/lesões , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Hepatocyte growth factor (HGF) is a pluripotent mitogen thought to be involved in liver regeneration. It is synthesized as a single chain promitogen and requires proteolytic processing to a two-chain heterodimeric form for biological activity. The pharmacokinetics and tissue distribution of radioiodinated single chain recombinant human HGF ([125I]rhuHGF) were studied in male Sprague-Dawley rats after an iv bolus dose. Pharmacokinetic parameters were determined from trichloroacetic acid-precipitable radioactivity in serum samples. There was a rapid distribution phase (t1/2 alpha = 3.1 min) and a slower elimination phase (t1/2 beta = 114 min). Tissue distribution was assessed by whole body autoradiography 5, 60, and 1440 min after an iv bolus dose. rhuHGF rapidly distributed to the liver, kidney, adrenal gland, and spleen. The importance of the liver in the rapid clearance and subsequent conversion of single chain pro-rhuHGF to the mitogenically active two-chain form was demonstrated using an isolated rat liver perfusion system. TCA-precipitable radioactivity excreted into the bile (1.0 +/- 0.1%) and released into the venous effluent (38.9 +/- 6.1%) was monitored for 60 min after a portal vein injection. The appearance of radioactivity in both the bile and venous effluent was maximal between 20-35 min. Further characterization of the reduced samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the two-chain form of [125I]rhuHGF was the predominant form after hepatic perfusion. These studies suggest that the liver plays a major role in the rapid clearance and subsequent activation of pro-rhuHGF in vivo.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/farmacocinética , Animais , Autorradiografia , Bile/metabolismo , Sangue/metabolismo , Células CHO , Cricetinae , Fibrinolisina/metabolismo , Fator de Crescimento de Hepatócito/química , Humanos , Técnicas In Vitro , Injeções Intravenosas , Radioisótopos do Iodo , Fígado/metabolismo , Masculino , Perfusão , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes , Distribuição TecidualRESUMO
Because examination of regulatory trans-phosphorylations can help elucidate the cellular functions of tyrosyl protein kinases, we have investigated the effects of phosphorylation by casein kinase-1 on the activity of the p40 tyrosyl protein kinase. We find that casein kinase-1 can phosphorylate the p40 tyrosyl kinase on serine and threonine residues, in part on a unique tryptic peptide. The phosphorylation induces a substantial increase in the tyrosyl protein kinase activity of p40, in contrast to most instances in which serine/threonine phosphorylation inhibits activity of tyrosyl protein kinases. These findings raise the possibility that p40 might be part of a protein phosphorylation network in which casein kinase-1 participates.
Assuntos
Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Caseína Quinases , Bovinos , Cromatografia Líquida de Alta Pressão , Ativação Enzimática , Peso Molecular , Fosfopeptídeos/isolamento & purificação , Fosforilação , Timo/enzimologiaRESUMO
A previous study has shown that complement component C3 binds to recombinant apolipoprotein(a) (r-apo(a)). In the present report we have investigated the interactions between lipoprotein(a) (Lp(a)), r-apo(a) and C3 in relation to complement activation and degradation. Neither Lp(a) nor r-apo(a) affected complement activation as indicated by sheep and rabbit red blood cell hemolytic assays, and by assessment of the amount of C3a generated in zymosan-activated human serum in the presence or absence of Lp(a). Crossed immunoelectrophoretic analyses indicated that Lp(a) retarded the migration of iC3b in complement-activated serum but had no effects on C3, C3b, C3c or C3dg. Recombinant apo(a) exhibited the same properties as intact Lp(a) indicating that it is the apo(a) portion of Lp(a) that mediates this effect and not the lipid moiety. Low density lipoprotein had no effect on the migration of C3 cleavage fragments. Treatment of Lp(a) or apo(a) with neuraminidase abolished their capacity to alter iC3b migration. SDS-PAGE immunoblotting analysis of C3 activation fragments generated in the presence of Lp(a) demonstrated the usual physiologic C3 cleavage fragments. Rocket intermediate gel immunoelectrophoresis of complement-activated serum demonstrated that Lp(a) did not hinder or accelerate the generation of C3c and C3dg breakdown fragments of iC3b. The results indicate that the apo(a) moiety of Lp(a) alters the migration of iC3b in an electric field but does not affect complement activation or degradation of activated C3. The sialic acid residues on apo(a) are necessary for the apo(a)-iC3b interaction.
Assuntos
Apolipoproteínas/metabolismo , Ativação do Complemento , Complemento C3/metabolismo , Lipoproteínas/fisiologia , Apolipoproteínas/química , Apoproteína(a) , Complemento C3b/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoeletroforese Bidimensional , Lipoproteína(a) , Lipoproteínas/metabolismo , Ácido N-Acetilneuramínico , Ácidos Siálicos/análiseRESUMO
BACKGROUND: Patients with severe myocardial ischemia who are not candidates for percutaneous or surgical revascularization have few therapeutic options. Therapeutic angiogenesis in animal models with use of recombinant human vascular endothelial growth factor (rhVEGF) has resulted in successful revascularization of ischemic myocardium. This was a dose escalation trial designed to determine the safety and tolerability of intracoronary rhVEGF infusions. METHODS AND RESULTS: Patients were eligible if they had stable exertional angina, a significant reversible perfusion defect by stress myocardial perfusion study, and coronary anatomy that was suboptimal for percutaneous coronary intervention or coronary artery bypass grafting. rhVEGF was administered to a total of 15 patients by 2 sequential (eg, right and left) intracoronary infusions, each for 10 minutes, at rates of 0.005 (n = 4), 0.017 (n = 4), 0.050 (n = 4), and 0.167 mg/kg/min (n = 3). Pharmacokinetic sampling and hemodynamic monitoring were performed for 24 hours. Radionuclide myocardial perfusion imaging was performed before treatment and at 30 and 60 days after treatment. Follow-up angiograms were performed on selected patients at 60 days. The maximally tolerated intracardiac dose of rhVEGF was 0.050 mg/kg/min. Minimal hemodynamic changes were seen at 0.0050 mg/kg/min (2% +/- 7% [SD] mean decrease in systolic blood pressure from baseline to nadir systolic blood pressure), whereas at 0.167 mg/kg/min there was a 28% +/- 7% mean decrease from baseline to nadir (136 to 95 mm Hg systolic). Myocardial perfusion imaging was improved in 7 of 14 patients at 60 days. All 7 patients with follow-up angiograms had improvements in the collateral density score. CONCLUSION: rhVEGF appears well tolerated by coronary infusion at rates up to 0.050 mg/kg/min. This study provides the basis for future clinical trials to assess the clinical benefit of therapeutic angiogenesis with rhVEGF.
Assuntos
Doença das Coronárias/tratamento farmacológico , Fatores de Crescimento Endotelial/administração & dosagem , Linfocinas/administração & dosagem , Isoformas de Proteínas/administração & dosagem , Circulação Coronária/efeitos dos fármacos , Fatores de Crescimento Endotelial/farmacologia , Fatores de Crescimento Endotelial/uso terapêutico , Humanos , Linfocinas/farmacologia , Linfocinas/uso terapêutico , Neovascularização Fisiológica/efeitos dos fármacos , Isoformas de Proteínas/farmacologia , Isoformas de Proteínas/uso terapêutico , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
A recombinant apo(a) containing 17 kringle 4 domains as well as the kringle 5 and protease domains of apo(a) was characterized by hydrodynamic studies and electron microscopy. Recombinant apo(a) is a monomer in solution with a molecular weight of 325,000 by sedimentation equilibrium and 320,000 by sedimentation and diffusion, and it is a highly asymmetric molecule with a frictional ratio of 2.2. In the electron microscope recombinant apo(a) is visualized as a flexible chain of domains approximately 800 A long. Sedimentation velocity studies also demonstrate that when it is mixed with LDL, recombinant apo(a) reversibly forms an Lp(a)-like complex with a 1:1 stoichiometry; moreover, complex formation is inhibited by 6-amino hexanoic acid. Hydrodynamic modeling and electron microscopy suggest that only a small portion of the r-apo(a) molecule interacts with the LDL and the rest of the chain extends into solution. Preliminary studies indicate that recombinant apo(a) also binds mouse LDL.
Assuntos
Apolipoproteínas/metabolismo , Apolipoproteínas/ultraestrutura , Lipoproteína(a) , Lipoproteínas LDL/metabolismo , Animais , Apolipoproteínas/química , Apoproteína(a) , Humanos , Técnicas In Vitro , Kringles , Camundongos , Microscopia Eletrônica , Peso Molecular , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Soluções , UltracentrifugaçãoRESUMO
A protein-tyrosine kinase has been isolated from a soluble extract of bovine thymus based on its ability to phosphorylate the tyrosine-containing peptide angiotensin I. The purification procedure employs sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-agarose, butyl-agarose, and Sephadex G-75. The purified enzyme (p40) is a monomer of Mr = 40,000. The p40 kinase contains an ATP-binding site as determined by photoaffinity labeling experiments and catalyzes an intramolecular autophosphorylation reaction that leads to its modification on tyrosine. Of several proteins tested only the cytoplasmic domain of the erythrocyte band 3 protein serves as a good substrate for p40 (Km = 12 microM). Increasing concentrations of NaCl stimulate the phosphorylation of angiotensin I, inhibit the phosphorylation of band 3, and have no effect on the autophosphorylation of p40. At low concentrations of NaCl, Mn2+ is the preferred divalent cation. Peptide mapping experiments indicate that p40 is distinct from pp60src and from the major phosphotyrosine containing proteins of T and B lymphocyte membranes.
Assuntos
Proteínas Tirosina Quinases/isolamento & purificação , Timo/enzimologia , Trifosfato de Adenosina/metabolismo , Angiotensina I/metabolismo , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Bovinos , Cromatografia , Linfócitos/enzimologia , Peso Molecular , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Cloreto de Sódio/farmacologia , Especificidade por SubstratoRESUMO
Tissue factor (TF) is an integral membrane glycoprotein that serves as a cellular receptor and cofactor for the activation of the plasma protease factor VII. TF activity in both monocytes and endothelial cells is regulated by various cytokines and mitogens, including the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). Three TF constructs (full-length human, a cytoplasmic domain deletion mutant, and a human-rat TF chimera), expressed in a human kidney cell line, were used to examine the in vivo phosphorylation state of TF after PMA treatment. The cytoplasmic domains of both rat and human TF were rapidly phosphorylated after cells were treated with 10-100 nM PMA. This response was completely abolished by preincubating cells with staurosporine, the potent PKC inhibitor, prior to PMA treatment. Localization of the phosphorylation site(s) to the cytoplasmic domain was demonstrated using a deletion mutant of TF and by CNBr digestion at the single methionine residue (Met-210) in the TF sequence. The rat TF cytoplasmic domain was phosphorylated to a higher specific activity than the human TF cytoplasmic domain. Phosphoamino acid analysis of the chimeric TF revealed both phosphothreonine and phosphoserine, whereas human TF contained only phosphoserine. Thus both potential phosphoacceptor sites are phosphorylated in the rat TF cytoplasmic domain. Alignment of TF cDNA sequences of mouse, rat, rabbit, and man revealed that the phosphoacceptor site (X-S*/T*-P-X, where asterisk indicates the phosphorylated residue) in the cytoplasmic domain has been conserved through evolution.
Assuntos
Proteína Quinase C/metabolismo , Tromboplastina/metabolismo , Alcaloides/farmacologia , Sequência de Aminoácidos , Linhagem Celular , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Rim/citologia , Rim/metabolismo , Dados de Sequência Molecular , Fosforilação , Testes de Precipitina , Proteína Quinase C/antagonistas & inibidores , Alinhamento de Sequência , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologia , Tromboplastina/genéticaRESUMO
The mechanism by which vascular endothelial growth factor (VEGF) regulates endothelial nitric-oxide synthase (eNOS) expression is presently unclear. Here we report that VEGF treatment of bovine adrenal cortex endothelial cells resulted in a 5-fold increase in both eNOS protein and activity. Endothelial NOS expression was maximal following 2 days of constant VEGF exposure (500 pM) and declined to base-line levels by day 5. The elevated eNOS protein level was sustained over the time course if VEGF was co-incubated with L-N(G)-nitroarginine methyl ester, a competitive eNOS inhibitor. Addition of S-nitroso-N-acetylpenicillamine, a nitric oxide donor, prevented VEGF-induced eNOS up-regulation. These data suggest that nitric oxide participates in a negative feedback mechanism regulating eNOS expression. Various approaches were used to investigate the role of the two high affinity VEGF receptors in eNOS up-regulation. A KDR receptor-selective mutant increased eNOS expression, whereas an Flt-1 receptor-selective mutant did not. Furthermore, VEGF treatment increased eNOS expression in a KDR but not in an Flt-1 receptor-transfected porcine aorta endothelial cell line. SU1498, a selective inhibitor of the KDR receptor tyrosine kinase, blocked eNOS up-regulation, thus providing further evidence that the KDR receptor signals for eNOS up-regulation. Finally, treatment of adrenal cortex endothelial cells with VEGF or phorbol ester resulted in protein kinase C activation and elevated eNOS expression, whereas inhibition of protein kinase C with isoform-specific inhibitors abolished VEGF-induced eNOS up-regulation. Taken together, these data demonstrate that VEGF increases eNOS expression via activation of the KDR receptor tyrosine kinase and a downstream protein kinase C signaling pathway.
Assuntos
Fatores de Crescimento Endotelial/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Linfocinas/farmacologia , Óxido Nítrico Sintase/metabolismo , Proteína Quinase C/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Córtex Suprarrenal , Animais , Bovinos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Substâncias de Crescimento/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo II , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes , Transdução de Sinais , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The major NaCl-stimulated protein-tyrosine kinase activity found in soluble thymus extracts, as measured by the phosphorylation of angiotensin I, is a 40-kDa enzyme known as p40 (Zioncheck, T. F., Harrison, M. L., and Geahlen, R. L. (1986) J. Biol. Chem. 261, 15637-15643). Antibodies prepared against p40 cross-react with a 72-kDa protein-tyrosine kinase (p72) from spleen or thymus that is closely related to p40 by peptide-mapping experiments. The recovery of p40 from spleen homogenates is reduced while the recovery of p72 is enhanced by the addition of high concentrations of leupeptin or soybean trypsin inhibitor to the homogenization media. The generation of p40 in spleen homogenates occurs with a concomitant increase in protein-tyrosine kinase activity. Activated catalytic fragments of 38-43 kDa can be generated by the treatment of partially purified p72 with trypsin or papain. The p72 protein-tyrosine kinase is found at the highest levels in spleen, thymus, and lung, tissues that also have high protein-tyrosine kinase activity and generate high levels of p40 following homogenization. p72 is also found in certain T and B cell-derived cell lines and in NIH3T3 cells.
Assuntos
Linfócitos/enzimologia , Proteínas Tirosina Quinases/sangue , Angiotensina I/metabolismo , Animais , Linhagem Celular , Isoenzimas/sangue , Peso Molecular , Mapeamento de Peptídeos , Fosforilação , Inibidores de Proteases/farmacologia , Cloreto de Sódio/farmacologiaRESUMO
Recombinant DNA-derived apolipoprotein(a) was used to demonstrate that the apo(a) moiety of lipoprotein(a) (Lp(a)) is responsible for the binding of Lp(a) to other apolipoprotein B-containing lipoproteins (apoB-Lp) including LDL2, a subclass of low density lipoproteins (d = 1.030-1.063 g/ml). The r-apo(a).LDL2 complexes exhibited the same binding constant as Lp(a).LDL2 (10(-8) M). Treatment of either recombinant apo(a) or Lp(a) with a reducing agent destroyed binding activity. A synthetic polypeptide corresponding to a portion of apo(a)'s kringle-4 inhibited the binding (K1 = 1.9 x 10(-4) M) of LDL2 to Lp(a). Therefore, we concluded that binding to apoB-Lp was mediated by the kringle-4-like domains on apo(a). Using ligand chromatography which can detect complexes having a KD as low as 10(-2) M, we demonstrated the binding of plasminogen to apoB-Lp. Like Lp(a), binding of plasminogen to apoB-Lp was mediated by the kringle domain(s). The differences in binding affinity may be due to amino acid substitutions in the kringle-4-like domain. In most of the kringle-4-like domains of apo(a), the aspartic residue critical for binding to lysine was substituted by valine. Consistent with this substitution, we found that L-proline and hydroxyproline, but not L-lysine, inhibited the binding of LDL2 to apo(a). Inhibition by L-proline could be reversed in the binding studies by increasing the amount of apo(a); and L-proline-Sepharose bound plasma Lp(a), suggesting that L-proline acted as a ligand for the kringle-4-like domain(s) of apo(a) involved in the binding of apoB-Lp. The binding of apo(a) to proline and hydroxyproline could be responsible for the binding of apo(a) to the subendothelial extracellular matrix, i.e. domains of proteins rich in proline or hydroxyproline (e.g. collagen and elastin).
Assuntos
Apolipoproteínas B/metabolismo , Apolipoproteínas/metabolismo , Lipoproteína(a) , Lipoproteínas/metabolismo , Aminoácidos/metabolismo , Animais , Apoproteína(a) , Ligação Competitiva , Western Blotting , Bovinos , Ditiotreitol , Eletroforese em Gel de Poliacrilamida , Imunodifusão , Fragmentos de Peptídeos/metabolismo , Plasminogênio/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
The receptor for hepatocyte growth factor (HGF) is the product of the c-met proto-oncogene, a membrane-spanning tyrosine kinase receptor. To facilitate analysis of HGF and its receptor (HGFr), we expressed and purified a chimeric protein containing the extracellular domain (ECD) of the HGFr fused to the constant region of IgG heavy chain. This soluble form of the HGFr (sHGFr) bound HGF with an affinity similar to that of the authentic, membrane-associated receptor. The sHGFr also neutralized the binding of HGF to the HGFr expressed on A549 cells. Like the mature form of the HGFr, sHGFr is a heterodimer which arises by proteolytic processing within the ECD. In order to characterize the requirements for proteolytic processing of the ECD and the effects of cleavage on ligand binding, we expressed sHGFr variants containing amino acid substitutions in the putative processing site. Replacement of the P1 or P4 arginine, but not the P3 lysine, with alanine inhibited conversion to the alpha/beta heterodimer. This suggests that maturation is mediated by furin or a furin-like protease. Finally, we showed that processing of the sHGFr into the alpha/beta form is not required for high affinity binding to either pro- or mature HGF.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Alanina , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA/genética , Biblioteca Gênica , Humanos , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Cinética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , Proteínas Tirosina Quinases/isolamento & purificação , Proteínas Tirosina Quinases/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas c-met , Proto-Oncogenes , Receptores de Superfície Celular/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas RecombinantesRESUMO
Endothelial cells express two related vascular endothelial growth factor (VEGF) receptor tyrosine kinases, KDR (kinase-insert domain containing receptor, or VEGFR-2) and Flt-1 (fms-like tyrosine kinase, or VEGFR-1). Although considerable experimental evidence links KDR activation to endothelial cell mitogenesis, there is still significant uncertainty concerning the role of individual VEGF receptors for other biological effects such as vascular permeability. VEGF mutants that bind to either KDR or Flt-1 with high selectivity were used to determine which of the two receptors serves to mediate different VEGF functions. In addition to mediating mitogenic signaling, selective KDR activation was sufficient for the activation of intracellular signaling pathways implicated in cell migration. KDR stimulation caused tyrosine phosphorylation of both phosphatidylinositol 3-kinase and phospholipase Cgamma in primary endothelial cells and stimulated cell migration. KDR-selective VEGF was also able to induce angiogenesis in the rat cornea to an extent indistinguishable from wild type VEGF. We also demonstrate that KDR, but not Flt-1, stimulation is responsible for the induction of vascular permeability by VEGF.
Assuntos
Endotélio Vascular/citologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Transdução de Sinais/fisiologia , Permeabilidade Capilar/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/fisiologia , Ativação Enzimática , Humanos , Isoenzimas/metabolismo , Linfocinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Neovascularização Fisiológica/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama , Receptores de Fatores de Crescimento do Endotélio Vascular , Fosfolipases Tipo C/metabolismo , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-alpha) have been shown to synergistically increase tissue factor (TF) expression in endothelial cells; however, the role of the VEGF receptors (KDR, Flt-1, and neuropilin) in this process is unclear. Here we report that VEGF binding to the KDR receptor is necessary and sufficient for the potentiation of TNF-induced TF expression in human umbilical vein endothelial cells. TF expression was evaluated by Western blot analysis and fluorescence-activated cell sorting. In the absence of TNF-alpha, wild-type VEGF- or KDR receptor-selective variants induced an approximate 7-fold increase in total TF expression. Treatment with TNF alone produced an approximate 110-fold increase in total TF expression, whereas coincubation of TNF-alpha with wild-type VEGF- or KDR-selective variants resulted in an approximate 250-fold increase in TF expression. VEGF lacking the heparin binding domain was also able to potentiate TF expression, indicating that heparin-sulfate proteoglycan or neuropilin binding is not required for TF up-regulation. Neither placental growth factor nor an Flt-1-selective variant was capable of inducing TF expression in the presence or absence of TNF. Inhibition of protein-tyrosine kinase or protein kinase C activity significantly blocked the TNF/VEGF potentiation of TF up-regulation, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, increased TF expression. These data demonstrate that KDR receptor signaling governs both VEGF-induced TF expression and the potentiation of TNF-induced up-regulation of TF.
Assuntos
Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/metabolismo , Linfocinas/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Tromboplastina/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Sinergismo Farmacológico , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Substâncias de Crescimento/farmacologia , Humanos , Linfocinas/genética , Mutação , Proteína Quinase C/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Estaurosporina/farmacologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We have stably expressed a recombinant form of apo(a) in a human embryonic kidney cell line. The engineered protein (predicted mass of 250 kDa) contains 17 copies of the apo(a) domain, which resembles kringle 4 of plasminogen, followed by the plasminogen-like kringle 5 and protease-like domain of apo(a). The recombinant protein [r-apo(a)] was isolated from cell culture media by immunoaffinity chromatography, and its physical properties were studied. As is the case for apo(a) isolated from plasma-derived Lp(a), r-apo(a) is highly glycosylated (23% by weight), containing both N- and O-linked glycans, which results in an observed molecular mass of 500 kDa by SDS-PAGE. The high sialic acid content was reflected in a pI of 4.3 for the r-apo(a). Two subpopulations of r-apo(a) secreted by the permanent cell line were identified with respect to lysine-Sepharose binding; the majority of the r-apo(a) bound specifically to this matrix and was eluted with epsilon-aminocaproic acid (epsilon-ACA). When the r-apo(a) plasmid was used to transfect a human hepatoma cell line, lipoprotein particles were secreted containing the disulfide-linked complex of apoB-100 and the r-apo(a). The density of these particles was shown to be heterogeneous, with the majority of the r-Lp(a) floating in the density range of plasma-derived Lp(a).
Assuntos
Apolipoproteínas/genética , Lipoproteína(a) , Apolipoproteínas/isolamento & purificação , Apolipoproteínas/metabolismo , Apoproteína(a) , Linhagem Celular , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Clonagem Molecular , Vetores Genéticos , Humanos , Peso Molecular , Plasmídeos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transfecção , Tunicamicina/farmacologiaRESUMO
We investigated the possibility that vascular endothelial growth factor (VEGF) treatment could regulate KDR/Flk-1 receptor expression in endothelial cells. Bovine adrenal cortex endothelial cells were incubated with 200 pM rhVEGF165 for 0-7 days. Western blot analysis showed a 3-5-fold increase in total KDR protein following 4-day VEGF treatment. Scatchard analysis revealed that VEGF induced a 2-3-fold increase in high affinity receptor number (5.0 x 10(4)/cell versus 2. 4 x 10(4)/cell) without significantly affecting receptor binding affinity (Kd 76 pM versus 72 pM). Quantitative polymerase chain reaction analysis demonstrated a 3-fold increase in KDR mRNA levels following VEGF exposure. VEGF-induced KDR expression primarily occurred at the transcriptional level as demonstrated by a luciferase reporter assay system. Receptor selective mutants with wild-type KDR binding and decreased Flt-1 binding also induced KDR up-regulation; in contrast, mutants with decreased KDR binding and wild-type Flt-1 binding did not, suggesting that KDR receptor signaling mediated the increase in KDR expression. Inhibition of tyrosine kinase, Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase activities all blocked VEGF-induced KDR up-regulation. Finally, co-incubation of nitric-oxide synthase inhibitors with VEGF had no significant effect on KDR expression, but 100 microM sodium nitroprusside, a NO donor, significantly inhibited VEGF-induced KDR up-regulation, indicating that NO negatively regulates KDR expression. In conclusion, our data demonstrate that VEGF binding to the KDR receptor tyrosine kinase results in an increase in KDR receptor gene transcription and protein expression. Thus, KDR up-regulation induced by VEGF may represent an important positive feedback mechanism for VEGF action in tumor and ischemia-induced angiogenesis.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Regulação para Cima , Animais , Anticorpos Monoclonais/imunologia , Bovinos , Células Cultivadas , Fatores de Crescimento Endotelial/imunologia , Linfocinas/imunologia , Testes de Neutralização , Óxido Nítrico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento do Endotélio Vascular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Recombinant apolipoprotein(a) has been studied by hydrodynamic techniques and electron microscopy. Recombinant apo(a) was primarily a monomer in solution with an s0(20,w) of 9.3 S, a D20,w of 2.29 ficks, and a molecular weight of 325,000 from sedimentation equilibrium and 318,000 from combining the sedimentation and diffusion coefficients. A small amount, approximately 10%, of the recombinant apo(a) was present as a high molecular weight aggregate. The Stokes radius of the monomer, determined either from the diffusion coefficient or by combining the sedimentation equilibrium data with the sedimentation velocity data, was 94 A. The frictional ratio was 2.2, suggesting a highly asymmetric or random coil structure. In the electron microscope, recombinant apolipoprotein(a) was visualized as a long, highly flexible chain of domains forming large, open coiled structures on the EM grid with contour lengths of about 800 A. Addition of 6-aminohexanoic acid at 50 mM, a concentration which should saturate the weak lysine binding sites, did not alter the sedimentation behavior. In vivo, apolipoprotein(a) is associated tightly with LDL to form a highly atherogenic lipoprotein, Lp(a). A single molecule of recombinant apo(a) also associated tightly with LDL to yield a 13.3-S Lp(a)-like complex. This complex dissociated upon the addition of 50 mM 6-aminohexanoic acid. A novel sucrose gradient centrifugation technique was employed to determine a dissociation constant for the reversible equilibrium between recombinant apo(a) and LDL; at physiological ionic strength the dissociation constant was 0.3 nM. Raising the salt concentration to 5 M NaBr caused the dissociation constant to increase to 500 nM.(ABSTRACT TRUNCATED AT 250 WORDS)