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1.
2.
The low density lipoprotein receptor-related protein 1 (LRP1) has been implicated in intracellular signaling functions as well as in lipid metabolism. Recent in vivo and in vitro studies suggest that LRP1 is a physiological modulator of the platelet-derived growth factor (PDGF) signaling pathway. Here we show that in mouse fibroblasts LRP1 modulates PDGF-BB signaling by controlling endocytosis and ligand-induced down-regulation of the PDGF receptor beta (PDGFRbeta). In LRP1-deficient fibroblasts, basal PDGFRbeta tyrosine kinase activity was derepressed, and PDGF-BB-induced endocytosis and degradation of PDGFRbeta were accelerated as compared with control cells. This was accompanied by rapid uptake of receptor-bound PDGF-BB into the cells and by attenuated ERK activation in response to PDGF-BB stimulation. Pulse-chase analysis indicated that the steady-state turnover rate of PDGFRbeta was also accelerated in LRP-deficient fibroblasts. The rapid degradation of PDGFRbeta in the LRP1-deficient fibroblasts was prevented by MG132 and chloroquine. Furthermore, the association of PDGFRbeta with c-Cbl, a ubiquitin E3-ligase, as well as the ligand-induced ubiquitination of PDGFRbeta were increased in LRP1-deficient fibroblasts. We show that LRP1 can directly interact with c-Cbl, suggesting a Sprouty-like role for LRP1 in regulating the access of the PDGFRbeta to the ubiquitination machinery. Thus, LRP1 modulates PDGF signaling by controlling ubiquitination and endocytosis of the PDGFRbeta. 相似文献
3.
It has been shown elsewhere that the epidermal growth factor (EGF) in A431 cells can recycle in receptor-bound state (Teslenko et al., 1987; Sorkin et al., 1989, 1991). Present study deals with the action of primaquine, a lysosomotropic agent, on EGF-receptor complexes (EGF-RC). By the method of indirect immunofluorescence with anti-EGF-R monoclonal antibody it is found that following a 1 h incubation of cells at 37 degrees C in the presence of EGF a bright staining of endosomes appears in the intranuclear region, while after incubation of the cells at 4 degrees only margins of cells are stained. Such a pattern of fluorescence is peculiar of endocytosis in A431 cells. When the cells were incubated in the presence of a 0.3 mM primaquine for 1 h, the immunostaining is changed: bright compact spot in the para-Golgi region appeared. The effect of primaquine is reversible. When the cells after preincubation with EGF were incubated in the absence of EGF for 3 h at 37 degrees C, the staining of cell margins could be observed again, demonstrating the recycling of EGF-RC. Under similar conditions of cell incubation, but in the presence of primaquine, the staining of the para-Golgi region was not changed. In the experiments with 125I-EGF it was shown that intracellular accumulations of 125I-EGF were maintained when the cells were incubated in the presence of 0.3 mM primaquine. It is concluded that primaquine inhibits the recycling of EGF-R in A431 cells. 相似文献
4.
A B-type PDGF receptor lacking most of the intracellular domain escapes degradation after ligand binding 总被引:5,自引:0,他引:5
The characteristics of the human B-type platelet-derived-growth-factor (PDGF) receptor expressed in Chinese hamster ovary (CHO) cells, were compared with those of a mutant receptor lacking all but 19 amino acids of the intracellular domain. The transfected wild-type receptor was synthesized as a 160-kDa precursor that was processed to 190 kDa. Each CHO cell expressed 30,000-100,000 receptors which bound PDGF-BB with a Kd of about 0.5 nM. Analysis of PDGF-AB binding yielded non-linear Scatchard plots; the major part of the binding sites had a Kd of 6 nM. PDGF-AA was not bound. The receptors expressed in CHO cells were down-regulated after binding of PDGF-BB, and mediated degradation of 125I-PDGF-BB with similar efficiency as PDGF-B-type receptors in human fibroblasts. The transfected receptor also transduced a mitogenic signal. The mutant receptor was synthesized as a 90-kDa precursor and was processed to 120 kDa with a slightly faster rate than the wild-type receptor. Cells expressing the mutant receptor generally had around 10(6) ligand-binding sites/cell, with a Kd for binding of PDGF-BB of 3 nM. The mutant receptor, which did not transduce a mitogenic response, mediated degradation of 125I-PDGF-BB, albeit less efficiently compared to the wild-type receptor. In contrast to the wild-type receptor, it was down-regulated only to a limited extent and not degraded in response to ligand binding. These findings indicate a role for the intracellular part of the receptor, not only in mitogenic signaling, but also in receptor internalization and intracellular routing. 相似文献
5.
Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction. 总被引:17,自引:4,他引:13
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L Severinsson B Ek K Mellstr?m L Claesson-Welsh C H Heldin 《Molecular and cellular biology》1990,10(2):801-809
A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor. 相似文献
6.
Rolny C Spillmann D Lindahl U Claesson-Welsh L 《The Journal of biological chemistry》2002,277(22):19315-19321
Platelet-derived growth factor (PDGF) induces mitogenic and migratory responses in a wide variety of cells, by activating specific receptor tyrosine kinases denoted the PDGF alpha- and beta-receptors. Different PDGF isoforms bind in a distinct manner to glycosaminoglycans, particularly heparan sulfate. In the present study, we show potentiation by exogenous heparin of PDGF-BB-induced PDGF alpha-receptor tyrosine phosphorylation in heparan sulfate-deficient Chinese hamster ovary (CHO) 677 cells. This effect was not seen for PDGF-AA treatment, and heparin lacked a potentiating effect on PDGF-BB stimulation of the PDGF beta-receptor. Heparin did not affect the affinity of PDGF-BB binding for the PDGF receptors on CHO 677 cells. The PDGF-BB-stimulated PDGF alpha-receptor phosphorylation was enhanced in a dose-dependent fashion by heparin at low concentration. The effect was modulated by 2-O- and 6-O-desulfation of the polysaccharide. Maximal induction of PDGF alpha-receptor tyrosine phosphorylation (6-fold) in CHO 677 cells was achieved by treatment with a heparin decasaccharide, but shorter oligosaccharides consisting of four or more monosaccharide units were also able to augment PDGF alpha-receptor phosphorylation, albeit at higher concentrations. Heparin potentiated PDGF-BB-induced activation of mitogen-activated protein kinase and protein kinase B (Akt) and allowed increased chemotaxis of the CHO 677 cells toward PDGF-BB. In conclusion, heparin modulates PDGF-BB-induced PDGF alpha-receptor phosphorylation and downstream signaling, with consequences for cellular responsiveness to the growth factor. 相似文献
7.
Functional analysis of aortic endothelial cells expressing mutant PDGF receptors with respect to expression of matrix metalloproteinase-3 总被引:2,自引:0,他引:2
Kanaki T Bujo H Mori S Yanjuan Z Takahashi K Yokote K Morisaki N Saito Y 《Biochemical and biophysical research communications》2002,294(2):231-237
Platelet-derived growth factor (PDGF) stimulates expression of matrix metalloproteinases (MMPs), including stromelysin-1 (MMP-3). Induction of these expressions is known to occur during the course of atherosclerosis, tumor invasion, and metastasis. We investigated PDGF-alpha receptor (alphaR)- and beta receptor (betaR)-mediated signaling pathways for the expression of MMP-3 and invasion activity using porcine aortic endothelial (PAE) cells with stable expression of normal or mutated PDGF receptors. RT-PCR and Western blot analyses revealed that PDGF-BB induces MMP-3 expression in PAE cells that exclusively express either the PDGF-alphaR or the -betaR, but not in non-transfected control cells. To identify the signals necessary for PDGF receptor-mediated induction of MMP-3 expression, several lines of PAE cells expressing mutant PDGF receptors were further analyzed. Cells expressing mutant PDGF receptors unable to associate with Src or PLCgamma, retained the ability to induce MMP-3 expression as a result of PDGF-BB stimulation. However, incubation with PDGF-BB did not induce MMP-3 expression in cells expressing a mutant PDGF-betaR unable to associate with phosphatidylinositol 3(')-kinase (PI3K). LY294002, a PI3K inhibitor, reduced PDGF-BB-stimulated MMP-3 expression in PAE cells expressing wild-type PDGF receptors. In contrast, PDGF-BB induced MMP-3 expression in the presence of U-73122, a PLCgamma inhibitor. Moreover, PDGF-BB enhanced the invasiveness of cells expressing wild type PDGF-beta receptors, but not of cells expressing mutant PDGF-betaRs impaired in their ability to associate with PI3K. In light of these results, it appears that PDGF-BB is capable of inducing MMP-3 expression through both the PDGF-alphaR and the -betaR, and the effects are contributed by the PI3K-mediated transduction pathways. 相似文献
8.
cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules. 总被引:48,自引:10,他引:38
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L Claesson-Welsh A Eriksson A Morén L Severinsson B Ek A Ostman C Betsholtz C H Heldin 《Molecular and cellular biology》1988,8(8):3476-3486
9.
Identification of the type-B receptor for platelet-derived growth factor in human embryonal carcinoma cells 总被引:2,自引:0,他引:2
S M Weima M A van Rooijen C L Mummery A Feyen S W de Laat E J van Zoelen 《Experimental cell research》1990,186(2):324-331
The human embryonal carcinoma (EC) cell line Tera 2 clone 13 (T2/13) can be induced to differentiate in vitro into neuroectodermal cell types with retinoic acid. Undifferentiated cells are characterized by rapid proliferation, whereas differentiated cells show a prolonged generation time, have a limited life span, and possess new cell-surface markers. In the present study we establish that both differentiated and undifferentiated T2/13 cells express the type-B platelet-derived growth factor (PDGF) receptor mRNA and bind PDGF-BB with high affinity. Differentiation causes a three-fold increase in receptor number per cell and leaves the affinity of the receptors unaffected. These data are the first to describe expression of this receptor in EC cells. The biosynthesis and degradation of this receptor were studied in undifferentiated as well as in differentiated T2/13 cells using an anti-type-B receptor antibody. These experiments revealed that high concentrations of recombinant PDGF-AA did not accelerate receptor metabolism in both cell types. In contrast, human PDGF or recombinant PDGF-BB added to the culture dishes readily increased receptor degradation. These results demonstrate that T2/13 cells express functional type-B PDGF receptors and suggest that cells responsive to PDGF might be present during mammalian development before the onset of mesoderm formation. 相似文献
10.
A Tingstr?m C Reuterdahl P Lindahl C H Heldin K Rubin 《Journal of immunology (Baltimore, Md. : 1950)》1992,148(2):546-554
Stimulation of human fibroblasts by platelet-derived growth factor (PDGF)-BB leads to a down-regulation of PDGF beta-receptors and a concomitant appearance of intracellular granular accumulations of receptors, as determined by stainings with the mAb PDGFR-B2. The granules contained both the ligand and PDGF beta-receptors, as revealed by double-immunofluorescence staining, and were formed in response to PDGF-BB but not in response to other cytokines tested. The formation of intracellular PDGF beta-receptor granules was dependent on PDGF-BB concentration and time of stimulation. The granular PDGF beta-receptor staining on cells treated with PDGF-BB for 1 h at 37 degrees C was used to investigate the effects of macrophage-derived cytokines on PDGF beta-receptor expression. The number of PDGF beta-receptor granules was found to be reduced in fibroblasts grown for 48 h in the presence of PDGF-BB, TNF-alpha, or IL-1; PDGF-AA under the same conditions had no effect. The reduction observed was paralleled by a decrease in cell surface expression of PDGF beta-receptors, measured as binding of 125I-PDGF-BB and of the PDGFR-B2 antibody. Furthermore, both TNF-alpha and IL-1 decreased the detergent-extractable pool of PDGF-beta receptors in the fibroblasts, as revealed by immunoblotting of detergent cell extracts. Finally, the decrease in PDGF beta-receptors after culturing of the cells in the presence of TNF-alpha and IL-1 was accompanied by a decreased incorporation of [3H]thymidine in response to PDGF-BB stimulation. In conclusion, our data suggest that certain macrophage-derived cytokines can modulate the expression of PDGF beta-receptors by cultured fibroblasts, which may contribute in part to their reduced responsiveness to PDGF. 相似文献
11.
《Journal of Lipid Mediators and Cell Signalling》1996,13(1):89-97
Platelet-derived growth factor (PDGF) is a potent mediator of fibroblast proliferation and chemotaxis. We have studied here the cytokine interferon-γ (IFN-γ) which is known to prime macrophages for increased PDGF production. Thus, we postulated that IFN-γ would act as a positive regulator of PDGF-BB secretion by rat alveolar macrophages, and in addition we asked whether or not the IFN-γ (a known anti-mitogenic cytokine) would block the growth response of primary lung fibroblasts to the PDGF-BB. Macrophages incubated with IFN-γ or iron spheres alone for 24 h secreted 2.5-fold more PDGF-BB than control macrophages incubated in serum-free medium. Preincubation of macrophages with IFN-γ prior to the addition of iron spheres synergistically increased PDGF-BB production 2–10-fold after 24 h. In contrast, when IFN-γ was added to quiescent rat lung fibroblasts (RLFs) in the presence of PDGF-BB, the cytokine induced a concentration-dependent decrease in cell growth, while IFN-γ alone did not affect proliferation. [125I]PDGF-BB receptor assays showed that neither preincubation nor coincubation of RLF with IFN-γ affected PDGF-BB binding to its receptors. 相似文献
12.
Effect of receptor kinase inactivation on the rate of internalization and degradation of PDGF and the PDGF beta-receptor 总被引:15,自引:3,他引:12
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The complementary DNAs for wildtype and tyrosine kinase-inactivated (K634A) forms of the PDGF beta-receptor were expressed in porcine aortic endothelial cells. We examined the internalization and degradation of ligands and receptors after exposure of receptor expressing cells to PDGF-BB, which binds to the beta-receptor with high affinity, and PDGF-AB, which binds with lower affinity. Cells expressing wildtype beta-receptors were able to internalize and degrade the receptor, as well as the ligand, after exposure to PDGF-BB or -AB. Cells expressing the kinase-inactivated mutant receptor also internalized and degraded both receptor and ligand, but with lower efficiency compared with the wildtype receptor cells. The degradation of either form of receptor was inhibited by treatment of the cells with the lysosomotropic drug chloroquine. Exposure of wildtype and K634A receptor expressing cells to PDGF-AB resulted in a twofold slower rate of internalization of this ligand as compared with PDGF-BB, whereas the relative rate of degradation was similar for the two ligands. Our data indicate that tyrosine kinase activity promotes, but is not a prerequisite for, ligand-induced internalization and degradation of the ligand-receptor complex. 相似文献
13.
14.
PDGF-AA and PDGF-BB biosynthesis: proprotein processing in the Golgi complex and lysosomal degradation of PDGF-BB retained intracellularly
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Platelet-derived growth factor is a potent mitogen for cells of mesenchymal origin. It is made up of two polypeptide chains (A and B) combined in three disulfide-linked dimeric forms (AA, AB, and BB). Here, the biosynthesis and proteolytic processing of the two homodimeric forms of PDGF (AA and BB) were studied in CHO cells stably transfected with A-chain (short splice version) or B-chain cDNA. PDGF-AA was processed to a 30-kD molecule which was secreted from the cells. In contrast, PDGF-BB formed two structurally distinct end products; a minor secreted 30-kD form and a major cell-associated 24-kD form. Immunocytochemical studies at light- and electron-microscopical levels revealed presence of PDGF in the Golgi complex, in lysosomes, and to a smaller extent in the ER. From analysis of cells treated with brefeldin A, an inhibitor of ER to Golgi transport, it was concluded that dimerization occurs in the ER, whereas the proteolytic processing of PDGF-AA and PDGF-BB precursors normally occurs in a compartment distal to the ER. Exposure of the cultures to the lysosomal inhibitor chloroquine led to an increased cellular accumulation of PDGF-BB, as determined both by metabolic labeling experiments and immunocytochemical methods, indicating that the retained form of PDGF-BB is normally degraded in lysosomes. Structural analysis of the two end products of PDGF-BB revealed that the secreted 30-kD form is a dimer of peptides processed as the B-chain of PDGF purified from human platelets, and that the retained 24-kD form is made up of subunits additionally processed in the NH2-terminus. Also, the 24-kD form was shown to be composed of proteolytic fragments held together by disulfide bridges. Taken together these findings suggest that the newly synthesized PDGF A- and B-chains are dimerized in the ER and thereafter transferred to the Golgi complex for proteolytic processing. From there, PDGF-AA is carried in vesicles to the cell surface for release extracellularly by exocytosis. A smaller part of PDGF-BB (the 30-kD form) is handled in a similar way, whereas the major part (the 24-kD form) is generated by additional proteolysis in the Golgi complex, from which it is slowly carried over to lysosomes for degradation. 相似文献
15.
Proteins derived from platelet alpha granules modulate the uptake of oxidized low density lipoprotein by macrophages. 总被引:2,自引:0,他引:2
Activated platelets secrete from their alpha granules a protein-like factor which stimulates the uptake of oxidized low-density lipoprotein (Ox-LDL) by macrophages. The aim of the present study was to evaluate the effect of three purified proteins obtained from platelet alpha granules: platelet-derived growth factor (PDGF), platelet factor-4 (PF-4), and beta-thromboglobulin (B-TG), on the uptake of Ox-LDL by macrophages. Cellular degradation of Ox-LDL by the J-774 A.1 macrophage-like cell line, that was preincubated for 18 h at 37 degrees C, with increasing concentrations of partially purified PDGF, (designated PDGF-CMS-III) was increased by up to 36% in comparison to control cells preincubated without PDGF. This effect was due to PDGF-mediated increase in the number of macrophage receptors for Ox-LDL. The enhanced uptake of Ox-LDL by PDGF resulted in an increase in cellular cholesterol content. Preincubation of macrophages with two types of recombinant PDGF dimers (10 ng/ml), revealed that PDGF-BB stimulated Ox-LDL cellular degradation by 64%, whereas PDGF-AB demonstrated only 34% stimulation, in comparison to control cells that were not treated with PDGF. The stimulatory effect of PDGF-CMS-III and PDGF-AB were reduced by 20% and 28%, respectively, when incubated in the presence of H-7, a specific protein kinase C inhibitor. When macrophages were preincubated with B-TG, cellular uptake of Ox-LDL was reduced by up to 30% at 100 ng B-TG/ml. This effect, however, was obtained only when B-TG was present in the incubation medium. Cellular degradation of Ox-LDL was not affected by preincubation of the cells with PF-4. Pretreatment of PCM with anti-PDGF or anti-B-TG antibodies abolished the effects of PCM on Ox-LDL degradation by macrophages. PDGF, thus, may represent the protein-like factor present in PCM which stimulates Ox-LDL degradation by macrophages, whereas B-TG may have a role in the recognition of PCM particles by the macrophage scavenger receptor. Modulation of macrophage cholesterol content by proteins secreted from activated platelets may have an important role in foam cell formation and atherosclerosis. 相似文献
16.
Platelet-derived growth factor (PDGF) is a dimeric molecule consisting of disulfide-bonded A- and B-polypeptide chains. Homodimeric (PDGF-AA, PDGF-BB) as well as heterodimeric (PDGF-AB) isoforms exert their effects on target cells by binding with different specificities to two structurally related protein tyrosine kinase receptors, denoted alpha- and beta-receptors. PDGF stimulates growth in various cell types, but little is known about its effect on mammalian cardiomyocytes. Therefore, growth-promoting effect of PDGF on rat cardiomyocytes was investigated. Primary culture of neonatal rat ventricular myocytes was prepared and cellular growth was estimated by [3H]-leucine incorporation assay. Tyrosine-phosphorylated PDGF-beta receptor of cardiomyocytes was determined by immunoblotting analysis after immunoprecipitation. PDGF-beta receptor, extracellular signal-regulated kinase (ERK) 1/2 and phosphorylated ERK1/2 of cardiomyocytes were measured by immunoblotting analysis. [3H]-leucine incorporation into the cultured myocytes was increased in a time- and dose-dependent manner after PDGF-BB stimulation. Phosphorylation of PDGF-beta receptor and ERK1/2 in cardiomyocytes was increased after short-term stimulation of PDGF-BB. Protein expression of PDGF-beta receptor and ERK1/2 was increased after long-term stimulation of PDGF-BB. [(3)H]-leucine incorporation into the cultured myocytes induced by PDGF-BB was partly blocked by mitogen-activated ERK-activating kinase (MEK) inhibitor PD98059, phospholipase C (PLC) inhibitor U73122, and protein kinase C (PKC) inhibitor staurosporin aglycone, respectively. Therefore, PDGF beta receptor, ERK1/2, PLC and PKC are involved in the signal transduction of PDGF-induced growth response of rat cardiac myocytes. 相似文献
17.
Alexander Sorkin Anders Eriksson Carl-Henrik Heldin Bengt Westermark Lena Claesson-Welsh 《Journal of cellular physiology》1993,156(2):373-382
We have examined the state of tyrosine phosphorylation of ligand-bound, internalized platelet-derived growth factor (PDGF) β-receptors. Analysis by immunofluorescence staining of cells stimulated with PDGF-BB at 370C indicated colocalization of phosphotyrosine, PDGF β-receptors, and PDGF-BB in endosome-like vesicles. Treatment of cells with an acidic buffer, which removed cell surfacebound PDGF-BB, led to a considerable decrease in phosphorylation and kinase activity of cell surface localized PDGF β-receptors, but not of internalized receptors. Immunoprecipitations using antisera against phosphotyrosine and the PDGF β-receptor from metabolically labeled cells showed that a major fraction of the tyrosine-phosphorylated pool of receptors were still accessible to the acid buffer treatment after 10 min of incubation of the cells at 370C. Under these conditions, about 20-25% of the total pool of tyrosine-phosphorylated, receptors were intratcellular, since they remained tyrosine phosphorylated after the acid buffer treatment. A considerable pool of tyrosine-phosphorylated, internalized receptors, after 10 min of incubation of the cells at 370C, could also be detected by immunoblotting analysis, using antisera against the PDGF β-receptor and phosphotyrosine. Analysis by in vitro kinase assays of immunoprecipitated PDGF β-receptors, obtained from PDGF-BB-stimulated cells different times after acid wash, showed that the internalized receptors retained kinase activity. These data suggest that a pool of internalized PDGF β-receptors remain active and may participate in signalling a considerable time after internalization. © 1993 Wiley-Liss, Inc. 相似文献
18.
PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex 总被引:115,自引:0,他引:115
Platelet-derived growth factor (PDGF) stimulated the tyrosine phosphorylation of the GTPase activating protein (GAP) in 3T3 cells and in CHO cells expressing wild-type PDGF receptors, but not in several CHO cell lines expressing mutant receptors defective in transmitting mitogenic signals. Following PDGF treatment of cells, GAP physically associated with the PDGF receptor and with Raf-1, phospholipase c-gamma, and PI-3 kinase, suggesting that PDGF induced the formation of complexes of signaling molecules. The association of GAP with the PDGF receptor and the phosphorylation of GAP with the PDGF receptor and the phosphorylation of GAP were reconstituted in vitro using purified protein and in insect cells expressing murine PDGF receptor and human GAP. However, in cells transformed by activated c-Ha-ras, which are defective in certain responses to PDGF, GAP failed to associate with the PDGF receptor or increase its phosphotyrosine content in response to PDGF. The association of GAP with ligand-activated PDGF receptors may directly link PDGF and ras signaling pathways. 相似文献
19.
The receptor tyrosine phosphatase (RPTP) LAR negatively regulates the activity of several receptor tyrosine kinases. To investigate if LAR affects the platelet-derived growth factor (PDGF) receptor signaling, mouse embryonic fibroblasts (MEFs) from mice where the LAR phosphatase domains were deleted (LARΔP), and wt littermates, were stimulated with 20 ng/ml PDGF-BB. In LAR phosphatase deficient MEFs, the phosphorylation of the PDGF β-receptor was surprisingly reduced, an event that was rescued by re-expression of wt LAR. The decreased phosphorylation of the PDGF β-receptor was observed independent of ligand concentration and occurred on all tyrosine residues, as determined by immunoblotting analysis using site-selective phosphotyrosine antibodies. This suggests that LAR is required for full PDGF β-receptor kinase activation. Downstream of receptor activation, phosphorylation of Akt and PLCγ were decreased in LARΔP MEFs, whereas Src and Erk MAP kinase pathways were less affected. The proliferation of LARΔP MEFs in response to PDGF-BB was also reduced. The inhibitory effect on the PDGF β-receptor in LARΔP cells was exerted via increased basal activity of c-Abl, since inhibition of c-Abl, by AG957 or siRNA, restored PDGF β-receptor phosphorylation. These observations suggest that LAR reduces the basal c-Abl activity thereby allowing for PDGF β-receptor kinase activation. 相似文献
20.
Differential expression of platelet-derived growth factor alpha- and beta- receptors on fat-storing cells and endothelial cells of rat liver. 总被引:5,自引:0,他引:5
P Heldin H Pertoft H Nordlinder C H Heldin T C Laurent 《Experimental cell research》1991,193(2):364-369
Fat-storing cells and endothelial cells of the liver sinusoids play important roles in the biosynthesis and degradation of hyaluronan (HYA). These cells were isolated from rat liver by a simple and rapid procedure involving: (1) cell separation by centrifugation on a Nycodenz gradient, after dispersion of the liver cells by collagenase perfusion; (2) further purification of the cells by centrifugation on a discontinuous Percoll gradient; and (3) culturing of the cells, taking advantage of the different time that seeded cells need for attachment to plastic surfaces. We have determined the effects of two isoforms of platelet-derived growth factor (PDGF), PDGF-BB and PDGF-AA, on HYA production by the original fat-storing cells, as well as by fat-storing cells which had changed in vitro to myofibroblast-like cells. PDGF-BB was found to stimulate HYA synthesis in both types of cells with a maximal response equal to that obtained with 10% fetal calf serum. PDGF-AA had no stimulatory effect on HYA production. Fat-storing cells and their modified myofibroblast-like phenotype bound specifically to 125I-PDGF-BB, but not to 125I-PDGF-AA, indicating that they had PDGF beta-receptors, but not alpha-receptors. In contrast, liver endothelial cells were found to have PDGF alpha-receptors, but not beta-receptors. 相似文献