Platelet-derived growth factor receptors form a high affinity state in membrane preparations. Kinetics and affinity cross-linking studies

The specific binding of 125I-PDGF (platelet-derived growth factor) to intact fibroblasts becomes relatively nondissociable during incubation at 37 degrees C. To characterize the interaction of PDGF with its receptors under conditions in which there is no receptor internalization, we have studied the binding of 125I-PDGF to membrane preparations derived from mouse 3T3 cells and rat liver. The binding sites had the affinity and specificity characteristics expected of PDGF receptors. At 37 degrees C (but not at 4 degrees C) the specific binding of 125I-PDGF to membranes gradually became nondissociable as assessed by either dilution or by addition of excess unlabeled PDGF. This tight binding was not due to a covalent interaction since the polyanionic compound suramin readily dissociated specifically bound 125I-PDGF. This property of suramin was used to expose rat liver PDGF receptors which were occupied by endogenous PDGF. Affinity cross-linking studies demonstrated that the formation of the nondissociable state of 125I-PDGF binding was associated with the binding of 125I-PDGF to a 160,000-dalton protein and to a 110,000-dalton species. The cross-linked binding sites could be adsorbed to wheat germ agglutinin and to anion exchange resins. The isoelectric point of both cross-linked species determined by two-dimensional gel electrophoresis was approximately 4.7. These data demonstrate that in membrane preparations, PDGF binds to an anionic 160,000-dalton glycoprotein which is likely to be the receptor. A high affinity state of PDGF binding, which is formed rapidly at 37 degrees C, can be dissociated by suramin.     

Characterization of the receptor for platelet-derived growth factor on human fibroblasts. Demonstration of an intimate relationship with a 185,000-Dalton substrate for the platelet-derived growth factor-stimulated kinase

The receptor for platelet-derived growth factor (PDGF) on human foreskin fibroblasts has been characterized. The molecular weight of the PDGF-receptor complex was estimated by affinity labeling techniques to about 200,000, as determined by sodium dodecyl sulfate-gel electrophoresis performed under reducing conditions. Subtraction of the Mr of reduced PDGF (18,000 to 15,000) gives a Mr for the receptor proper of 185,000 (+/- 10,000). The mobility in sodium dodecyl sulfate-gel electrophoresis was similar whether or not reducing agents were present, suggesting that the receptor may be a single chain protein. The hydrodynamic size of the 125I-PDGF-receptor complex after solubilization with Triton X-100, corresponded to a Mr of approximately 320,000, as determined by gel chromatography. Subtraction of the Mr contributions from Triton X-100 and PDGF, respectively, gives a Mr of approximately 200,000 for the receptor itself, an estimate in good agreement with the value obtained from the affinity-labeling experiments. Several lectins were analyzed for their ability to inhibit binding of 125I-PDGF to its receptor. It was found that wheat germ agglutinin and a lectin from Crotalaria juncea were effective inhibitors and that their inhibitory effects could be neutralized by N-acetylglucosamine and galactose, respectively, suggesting that the receptor contains these sugars. The properties of the receptor were compared with those of a 185,000-Da component, being the major substrate for the membrane-bound PDGF-stimulated kinase. It was found that the 185,000-Da component behaved similar to the PDGF receptor in sodium dodecyl sulfate-gel electrophoresis, performed with or without reducing agents present. Further, the 185,000-Da component co-eluted with the PDGF receptor on a Sepharose 6B column, and had affinity for the same lectins that inhibited the binding of 125I-PDGF to its receptor. Finally, the 185,000-Da component had affinity for PDGF immobilized on Sepharose beads, suggesting that it has PDGF-binding activity. We conclude that the PDGF receptor and the 185,000-Da substrate for the PDGF-dependent kinase are intimately related and probably identical molecules.     

Platelet-derived growth factor. II. Specific binding to cultured cells

We have prepared radioiodinated purified platelet-derived growth factor (125I-PDGF) which retains full mitogenic activity. The binding of 125I-PDGF to Swiss 3T3 cells is saturable and highly competed by whole blood serum, purified unlabeled PDGF, and by material from each stage in the purification of PDGF from platelet-rich plasma. Other purified mitogens and substances tested do not compete. 125I-PDGF binding to fibroblasts, 3T3 cells, and arterial smooth muscle cells shows an apparent dissociation constant of 10(-11) M, comparable to the range in which PDGF is mitogenic. A clone of Swiss 3T3 cells obtained from a population selected repeatedly against mitogenic response to PDGF shows a greatly reduced mitogenic response to PDGF and binds only 5% as much 125I-PDGF/cell. The binding capacity of the different cell types tested ranges from 2,500 binding sites/cell on the poorly responding variant to 390,000 binding sites/cell on one strain of Swiss 3T3 cells. Cell types that do not respond to PDGF do not show specific high affinity binding of 125I-PDGF. At 4 degrees C, 125I-PDGF binding to monolayer cultures is relatively slow. Equilibrium binding of low concentrations of 125I-PDGF is not achieved during 7 h unless the binding medium is constantly mixed. 125I-PDGF binding at 4 degrees C shows a broad pH optimum between 6.3 and 8.0. Binding does not seem to require Ca2+ or Mg2+ but is reduced more than 6-fold if both monovalent and divalent salts are omitted. The initial rate of 125I-PDGF binding is greater at 37 degrees C than at 4 degrees C but cell-associated 125I begins to decline soon after reaching a peak value at 30-60 min. Coincident with this decline, trichloroacetic acid-soluble 125I appears in the medium and the binding capacity of the cells declines. These phenomena suggest that PDGF and its receptor may be internalized and degraded.     

Interactions between the receptors for platelet-derived growth factor and epidermal growth factor

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.     

Suramin binds to platelet-derived growth factor and inhibits its biological activity

The polyanion suramin was recently found to inhibit binding of 125I-PDGF (platelet-derived growth factor) to Balb/c 3T3 cell membranes. Cultured Swiss 3T3 cells were used to investigate the mode of action of suramin and to monitor its effect on the biological activity of PDGF. Evidence is presented that suramin inhibits cellular binding of PDGF by binding to PDGF itself, thereby preventing it from binding to its cell surface receptor: First, while suramin inhibited 125I-PDGF binding with a half maximum inhibition concentration of approximately 60 microM or 90 micrograms/ml in a simultaneous competition assay, it was inactive in a sequential radioreceptor assay, in which an inhibitor is expected to be active if it interacts with the receptor (even with relatively low affinity) but to be inactive if it interacts with PDGF. Second, suramin prevented immunoprecipitation of 125I-PDGF in a dose-dependent manner, with a half maximum effective concentration of approximately 50 microM. Furthermore, suramin efficiently dissociated 125I-PDGF bound to its cell surface receptor, whereas unlabeled PDGF even in large excess was virtually inactive. This is also in line with the proposed direct interaction between PDGF and suramin, since such an interaction can be envisaged to induce a conformational change in the PDGF-receptor complex, resulting in an increased off-rate of the complex. Reduced 125I-PDGF binding in the presence of suramin correlated directly with a suramin dose-dependent inhibition of PDGF-induced incorporation of 3H-thymidine into quiescent Swiss 3T3 cells and of the proliferation of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protamine inhibits platelet derived growth factor receptor activity but not epidermal growth factor activity

Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NP1 cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrosine specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW approximately 11,000 daltons and 7,000 daltons, respectively) is approximately 0.4 microM. Protamine II (MW approximately 4,800 daltons) was equally active (half maximum inhibition concentration approximately 0.4 microM); protamine IV (MW approximately 3,300 daltons) was substantially less active (half maximum inhibition concentration approximately 2.8 microM). These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells.     

Molecular characteristics of nerve growth factor receptors on PC12 cells

Cross-linking of 125I-nerve growth factor (NGF) to PC12 cells with the photoreactive heterobifunctional agent N-hydroxysuccinimidyl-4-azidobenzoate results in the labeling of two major bands with Mr 158,000 and 100,000 and a minor band with Mr 225,000 as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions. Binding of 125I-NGF to and cross-linking into all these species is abolished in the presence of excess unlabeled NGF but not in the presence of unlabeled epidermal growth factor, insulin, or bovine pancreatic trypsin inhibitor. When PC12 cells with bound 125I-NGF are incubated in excess unlabeled NGF at 0 degree C prior to cross-linking, only the Mr 158,000 species remains. In addition, binding of 125I-NGF to the Mr 158,000 complex is trypsin-resistant, whereas binding to the Mr 100,000 complex is not. These experiments identify the Mr 158,000 species as the slow NGF-receptor complex (chase stable at 0 degree C) and the smaller Mr 100,000 species as the fast NGF-receptor complex (trypsin sensitive). Furthermore, 125I-NGF bound to the former but not to the latter species is displaced by very-low concentrations of NGF, showing that at least a significant fraction of the high-molecular-weight slow receptor is also a high-affinity receptor. This identification is supported by the finding that chick sensory neurons which possess both high- and low-affinity receptors exhibit two major labeled bands with Mr 145,000 and 105,000 as a result of cross-linking with 125I-NGF, whereas a cell population enriched in non-neuronal cells, which possess only low-affinity receptors, exhibits only the Mr 105,000 component. A shift in molecular weight of both species after pretreatment with neuraminidase indicates that both complexes contain sialoglycoproteins and rules out the possibility that differences in sialic acid content are responsible for the difference in molecular weight of the two complexes. The relative amount of the labeling of these two complexes is not affected by the presence of protease inhibitors nor by a variation of 5000-fold in cross-linker concentration. These results place some limits on possible models for the NGF receptors and their interconversion.     

Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.

Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell mitogen which stimulates angiogenesis. Here we report that a previously identified receptor tyrosine kinase gene, KDR, encodes a receptor for VEGF. Expression of KDR in CMT-3 (cells which do not contain receptors for VEGF) allows for saturable 125I-VEGF binding with high affinity (KD = 75 pM). Affinity cross-linking of 125I-VEGF to KDR-transfected CMT-3 cells results in specific labeling of two proteins of M(r) = 195 and 235 kDa. The KDR receptor tyrosine kinase shares structural similarities with a recently reported receptor for VEGF, flt, in a manner reminiscent of the similarities between the alpha and beta forms of the PDGF receptors.     

Characterization of two monoclonal antibodies reactive with the external domain of the platelet-derived growth factor receptor

Two monoclonal antibodies against the receptor for platelet-derived growth factor (PDGF) were obtained by immunizing mice with pure PDGF receptor preparations derived from porcine uterus. The antibodies, denoted PDGFR-B1 and PDGFR-B2, both bound to the external domain of the receptor, as demonstrated by indirect immunofluorescence and binding of 125I-labeled antibodies to intact human fibroblasts. Both antibodies precipitated pure 175-kDa 32P-labeled autophosphorylated porcine PDGF receptor as well as a Mr 175,000 glycoprotein from metabolically labeled cells. The monoclonal antibodies did not inhibit binding of 125I-PDGF to human fibroblasts and did not stimulate these cells to undergo mitosis. Both antibodies induced clustering and down-regulation of their antigen. However, this resulted in only a partial loss of cell surface binding sites for PDGF itself, consistent with the conclusion that the monoclonals recognized only one of two or several receptors for PDGF. Clustering and down-regulation were not seen when the cells were incubated with monovalent Fab' fragments of the PDGFR-B2 antibody. The antibodies also stimulated autophosphorylation of pure PDGF receptor, and PDGFR-B2 was shown to stimulate phosphorylation of phosphofructokinase, an exogenous substrate for the PDGF receptor kinase. High concentrations of PDGFR-B2 antibody, or Fab' fragments thereof, failed to enhance the PDGF receptor kinase activity, compatible with the possibility that dimerization was of importance in the antibody-stimulated kinase activity of purified PDGF receptors.     

Characterization of a neuronal subtype of insulin-like growth factor I receptor

Primary neuronal cultures from fetal rat brain were utilized to investigate the possible role of insulin-like growth factor I (IGF-I) in neuronal growth and differentiation. 125I-IGF-I binding to intact cultured neurons was specific and saturable with an apparent Kd of 7.0 +/- 1.2 nM and a Bmax of 1.8 +/- 0.3 pmol/mg protein. Binding of 125I-IGF-I to neurons was inhibited by IGF-I, followed by IGF-II and insulin. 7 S nerve growth factor, but not beta-nerve growth factor, also inhibited 125I-IGF-I binding. A similar binding site was detected on brain membranes. Affinity cross-linking of 125I-IGF-I to intact cultured neurons revealed, under reducing conditions, a major binding moiety with an Mr of 115,000 and a minor component at Mr 260,000. The former represents a neuronal type of the IGF-I receptor alpha subunit, whereas the latter probably represents an alpha dimer. The Mr = 115,000 binding component for 125I-IGF-I was also present in membranes prepared from postnatal whole brain. In contrast, the binding moiety in cultured glial cells was of Mr = 135,000, which was identical to the IGF-I receptor alpha subunit of placenta. Thus mature brain, despite its cellular heterogeneity, expresses a structural subtype of IGF-I receptor which appears to be unique to differentiated neurons. Moreover, glial and neuronal cultures secreted a polypeptide which specifically bound IGF-I; the apparent Mr of this binding protein was determined by affinity cross-linking to be approximately 35,000. The presence of neuronal IGF-I receptors and binding proteins suggested that IGF-I may exert neurotrophic effects on developing neurons. This possibility was supported by the observation that IGF-I markedly stimulated neuronal RNA synthesis.     

Tumor promoter enhances mitogenesis by PDGF with little effect on PDGF binding

Preincubation of Swiss 3T3 cells with the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) at 37 degrees C is observed to cause only a small (approximately 10%) decrease in maximal binding of 125I-platelet-derived growth factor (125I-PDGF), and does not affect the affinity of 125I-PDGF binding to these cells. Under the same conditions, the affinity of the epidermal growth factor receptor is greatly reduced, possibly resulting from phosphorylation by protein kinase C. TPA is also shown to have no effect on the kinetics of internalization or degradation of bound 125I-PDGF. Although TPA has little or no effect on these properties of the PDGF receptor, it was found to act in a synergistic fashion with low, but not high, concentrations of PDGF to increase DNA synthesis by 3T3 cells. Since TPA has previously been shown to activate protein kinase C, these findings suggest that protein kinase C does not regulate the ligand-binding properties of the PDGF receptor, and that the observed synergism between TPA and PDGF in stimulating mitogenesis reflects effects of TPA on other processes in the mitogenic pathway.     

Effects of platelet-derived growth factor on phosphorylation of the epidermal growth factor receptor in human skin fibroblasts

Heterologous regulation of the epidermal growth factor (EGF) receptor by platelet-derived growth factor (PDGF) was studied in FS4 human skin fibroblasts. The addition of PDGF to FS4 cells inhibited high affinity binding of 125I-EGF and stimulated phosphorylation of the EGF receptor. Phosphopeptide analysis by high performance liquid chromatography revealed that PDGF treatment of cells increased phosphorylation at several distinct sites of the EGF receptor. However, PDGF did not stimulate phosphorylation of threonine 654, a residue previously shown to be phosphorylated when protein kinase C is activated. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also stimulated phosphorylation of the same peptides from the EGF receptor as PDGF, and, in addition, induced phosphorylation of threonine 654. TPA inhibited both high and low affinity 125I-EGF binding by these cells. PDGF treatment of cells had no effect on EGF-dependent, tyrosine-specific autophosphorylation of the receptor, whereas TPA treatment was inhibitory. TPA, but not PDGF, stimulated phosphorylation of a Mr = 80,000 protein, known to be a substrate for protein kinase C, even though PDGF appeared to mediate breakdown of phosphoinositides. These data suggest that regulation of EGF receptor function by PDGF and TPA are distinct in these cells, even though some elements of regulation are shared. The results differ from those previously reported for a human lung fibroblast isolate, indicating that cell type-specific differences may exist in metabolism of the EGF receptor.     

Kinetics of 125I-PDGF binding and down-regulation of PDGF receptor in human arterial smooth muscle cell strains during cellular senescence in vitro

Platelet-derived growth factor (PDGF) is one of the major mitogens in serum to stimulate replication of human smooth muscle cells (SMCs) in culture. Previous studies using human fibroblasts failed to demonstrate changes in the receptor systems for growth factors during cellular senescence. We investigated the kinetics of ¹²⁵I-PDGF(-BB) binding and down-regulation of the PDGF receptor in three human arterial SMC strains during cellular aging. The number of specific ¹²⁵I-PDGF binding sites per cell increased slightly at a population doubling level (PDL) of 60%–80% of life span and then decreased at the PDL above 90%. The number of receptors per cell-surface area decreased with increasing in vitro age. The apparent Kd for the ¹²⁵I-PDGF binding decreased with in vitro senescence. The internalization and degradation of ¹²⁵I-PDGF per receptor were significantly reduced in senescent SMCs than young cells. Furthermore, down-regulation of the PDGF receptor was significantly greater in sensescent SMCs than young cells. Immunoblot studies demonstrated that changes in b?-subunit of the PDGF receptor accounted for those in the studies using ¹²⁵I-PDGF and that tyrosine phosphorylation of the PDGF receptor was significantly greater in young SMCs than aged cells. Our results suggest that age-related changes in the receptor systems for PDGF may be important contributors to the failure of DNA synthesis in senescent SMCs. © 1995 Wiley-Liss, Inc.     

Ligand-induced dimerization of the platelet-derived growth factor receptor. Monomer-dimer interconversion occurs independent of receptor phosphorylation

The platelet-derived growth factor (PDGF) receptor is a single membrane-spanning polypeptide of 180,000 daltons with a ligand-stimulatable tyrosine kinase site. We have investigated changes in the structure and association state of the receptor that are induced by ligand binding, but which precede autophosphorylation. Chemical cross-linking of PDGF-bound 32P-labeled receptor and 125I-PDGF-labeled receptor resulted in the generation of a radiolabeled cross-linked complex of 370-390 kDa. This band, as well as the 180-190-kDa PDGF receptor band, were recognized by a PDGF receptor-specific antipeptide antibody. The appearance of the 370-390-kDa band was PDGF-dependent and was seen irrespective of whether the receptor was membrane-bound, solubilized, or highly (approximately 90%) purified. Sedimentation analysis of the 125I-PDGF cross-linked receptor showed that both 180-190- and 370-390-kDa labeled species sedimented as a single peak at about 11.5 S, a position expected of a receptor dimer, demonstrating that the liganded receptor exists essentially as a dimer. In contrast, unliganded receptors sedimented as a single species at 7 S, a position consistent with a monomeric structure. The monomer-dimer interconversion was absolutely ligand-dependent and occurred independent of autophosphorylation. These results demonstrate and intimate correlation between PDGF binding and inter-receptor bond formation, and raise the possibility that the phenomenon may be causally linked to the process of kinase activation.     

Characterization of a Platelet-Derived Growth Factor Receptor on Swiss 3T3 Cells by Affinity Crosslinking

Abstract

Crosslinking experiments with various bifunctional reagents were used to investigate the nature and fate of the platelet growth factor (PDGF) receptor on Swiss mouse 3T3 cells. With ethylene glycol bis succinimidyl succinate (EGS) two bands with Mr 205′000 and Mr 190′000 were labeled at equal intensity, while with disuccinimidyl suberate (DSS) and the photoactivatable pazidophenylglyoxal (pAPG) almost exclusively the latter band was labeled, when analyzed by SDS polyacrylamide gel electrophoresis under reducing conditions. Evidence is presented that the Mr 190′000 band represents a Mr 175′000 receptor protein crosslinked to a single chain of the PDGF-dimer and the Mr 205′000 species the same Mr 175′000 protein crosslinked to both chains of PDGF. Pretreatment of cells with tunicamycin generated a third labeled band with Mr 150′000, while pretreatment with neuraminidase resulted in a shift of the Mr 205′000 and 190′000 bands by 5′000. This shows that the PDGF receptor is a sialoglycoprotein, consisting of a Mr β 135′000 proteinaceous core and a Mr β 40′000 carbohydrate moiety containing sialic acid. The virtually unchanged labeling intensity seen with tunicamycin and neuraminidase pretreated cells further suggests that the carbohydrate portion of the receptor is not required for PDGF binding. Finally, the crosslinking technique was used to show that at 37°C preformed ¹²⁵I-PDGF receptor complexes disappear from the cell surface with a t_1/2 β 8 min.     

Dissociation of the ligand and dephosphorylation of the platelet-derived growth factor receptor

The ligand-induced phosphorylation of the platelet-derived growth factor (PDGF) receptor was followed at 37 degrees C by a rapid dephosphorylation which was roughly parallel to the down regulation of the 125I-PDGF binding sites. At 4 degrees C, when the ligand-receptor complexes remain associated with the cell surface, the phosphorylated form of the receptor was more stable. However if the ligand was dissociated from the receptor by means of a mild acid wash or a treatment with suramin, the dephosphorylation of the receptor also occurred at a low temperature. These data suggest that, due to the dissociation of the ligand, the kinase activity of the receptor is switched off so that the phosphotyrosine-containing receptors remain exposed to the action of phosphatases that rapidly dephosphorylate them.     

PDGF BB stimulates proliferation and differentiation in cultured chondrocytes from rat rib growth plate.

The role of two isoforms (PDGF AA and PDGF BB) of platelet derived growth factor either alone or in combination with insulin-like growth factor I, on the regulation of proliferation and differentiation of rat rib growth plate chondrocytes was analyzed. PDGF BB increased DNA-synthesis in a dose dependent manner with a half maximal effect at 1 ng/ml. When PDGF BB was combined with IGF-I, an additive effect on DNA-synthesis was observed. PDGF AA and BB alone or combined with IGF-I had no appreciable effects on proteoglycan synthesis. Both homodimers caused an increase in AP-activity, indicating stimulation of cell differentiation. Cultured chondrocytes bound 125I-PDGF AA and 125I-PDGF BB and after stimulation with PDGF expressed c-fos protein. Thus, both homodimers play an important role in chondrocyte differentiation and together with IGF-I interact in the regulation of longitudinal bone growth.     

Basic fibroblast growth factor modulates the mitogenic potency of the platelet-derived growth factor (PDGF) isoforms by specific upregulation of the PDGF alpha receptor in vascular smooth muscle cells.

Platelet-derived growth factor AA (PDGF AA), in contrast to PDGF AB and BB, is a poor mitogen for smooth muscle cells (SMC). However, together with basic fibroblast growth factor (bFGF) it acts synergistically on DNA synthesis of these cells. Northern blot analysis revealed that bFGF selectively increases the PDGF-receptor alpha subtype (PDGF-R alpha) mRNA level without a significant effect on the PDGF-R beta mRNA level. The amount of PDGF-R alpha protein is also selectively increased after stimulating SMC with bFGF as shown by immunoprecipitation of lysates from SMC with anti-PDGF-R alpha antibodies. The number of binding sites for 125I-PDGF AA is more than doubled after bFGF-treatment, whereas the specific binding for PDGF AB and BB increased only by approximately 30 and 20%, respectively. The increase in the number of PDGF-R alpha renders the SMC responsive for PDGF AA as demonstrated by the induction of the proto-oncogene c-fos as well as by an increased cell proliferation. The enhanced PDGF binding after bFGF treatment may in fact explain the observed synergistic behavior. These data are discussed with regard to a possible role of growth factor-induced transmodulation of receptor expression during atherogenesis.     

Similar effects of platelet-derived growth factor and epidermal growth factor on the phosphorylation of tyrosine in cellular proteins

Platelet-derived growth factor (PDGF) stimulates the phosphorylation of proteins at tyrosine when added to quiescent 3T3 cells, as evidenced by the increase in the amount of phosphotyrosine, relative to phosphoserine and phosphothreonine, in cellular proteins. The increase was detected within 1 min of adding PDGF and was maximal by 5 min. This effect showed the same dependence on PDGF concentration as did association of 125I-PDGF with the cells. In different 3T3 cell lines the magnitude of the increase was approximately proportional to the number of PDGF receptors per cell. A number of proteins phosphorylated at tyrosine in response to PDGF have been detected by two-dimensional gel electrophoresis. They include a pair of related 45 kilodalton phosphoproteins, a pair of related 43 kilodalton phosphoproteins and a 42 kilodalton phosphoprotein. Similar changes were noted when quiescent 3T3 cells were incubated with epidermal growth factor. Possibly, these phosphoproteins are primary substrates of the tyrosine protein kinases activated by the receptors for PDGF and epidermal growth factor, and are involved in physiological effects common to the two growth factors.