A B-type PDGF receptor lacking most of the intracellular domain escapes degradation after ligand binding

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.     

Receptor binding of PDGF-AA and PDGF-BB,and the modulation of PDGF receptors by TGF-β, in human periodontal ligament cells

The growth factors PDGF-AA and PDGF-BB have previously been shown to be potent mitogens for human periodontal ligament (hPDL) cells in vitro. Additionally, the mitogenic response to PDGF-AA has been shown to be specifically inhibited by TGF-β. The purpose of the present investigation was to examine the binding of PDGF-AA and PDGF-BB, and the modulation of PDGF binding by TGF-β, in hPDL cells. Scatchard analysis identified an average of 32,000 PDGF-AA high-affinity binding sites per cell with a dissociation constant (K_d) of 0.66 nM and an average of 36,000 PDGF-BB binding sites per cell with a dissociation constant (k_d) of 0.44 nM. After treatment with TGF-β, the receptor number for PDGF-AA was found to specifically decrease by approximately 50%, with no change in binding affinity. This reduced number of binding sites was shown to correlate with both a decrease in levels of receptor tyrosine phosphorylation and a decreased number of α receptor subunits. Northern blot analysis identified the TGF-β-mediated decrease in PDGF α receptor subunit mRNA levels. PDGF-BB showed little change in the number of binding sites or in the binding affinity with TGF-β treatment, and the data were consistent with an increase in the number of β receptor subunits. These results demonstrate nearly equivalent numbers of receptors for both PDGF-AA and PDGF-BB in hPDL cells. Also, modulation of PDGF binding, by TFG-β, was shown to result in a reduced number of α receptor subunits with an increase in the number of β receptor subunits. © 1995 Wiley-Liss, Inc.     

Autophosphorylation within insulin receptor beta-subunits can occur as an intramolecular process.

The insulin receptor is a complex membrane-spanning glycoprotein composed of two alpha-subunits and two beta-subunits connected to form an alpha 2 beta 2 holoreceptor. Insulin binding to the extracellular alpha-subunits activates intracellular beta-subunit autophosphorylation and substrate kinase activity. The current study was designed to differentiate mechanisms of transmembrane signaling by the insulin receptor, specifically whether individual beta-subunits undergo cis- or trans-phosphorylation. We compared relative kinase activities of trypsin-truncated receptors, alpha beta-half receptors, and alpha 2 beta 2 holoreceptors under conditions that allowed us to differentiate intermolecular and intramolecular events. Compared to the insulin-stimulated holoreceptors, the trypsin-truncated receptor undergoes autophosphorylation at similar tyrosine residues and catalyzes substrate phosphorylation in the absence of insulin at a comparable rate. The truncated receptor sediments on a sucrose gradient at a position consistent with a structure comprising a single beta-subunit attached to a fragment of the alpha-subunit and undergoes autophosphorylation in this form in the absence of insulin. Autophosphorylation of the truncated insulin receptor is independent of receptor concentration, and immobilization of the truncated receptor on a matrix composed of an anti-receptor antibody bound to protein A-Sepharose diminishes neither autophosphorylation nor receptor-catalyzed substrate phosphorylation. Therefore, true intramolecular (cis) phosphorylations, which occur within individual beta-subunits derived from trypsin-truncated receptors, lead to kinase activation. However, insulin-stimulated autophosphorylation of insulin receptor alpha beta heterodimers is concentration-dependent, and both autophosphorylation and kinase activity are markedly reduced following immobilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparin amplifies platelet-derived growth factor (PDGF)- BB-induced PDGF alpha -receptor but not PDGF beta -receptor tyrosine phosphorylation in heparan sulfate-deficient cells. Effects on signal transduction and biological responses

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.     

Identification of two juxtamembrane autophosphorylation sites in the PDGF beta-receptor; involvement in the interaction with Src family tyrosine kinases.

Two novel sites of autophosphorylation were localized to the juxtamembrane segment of the human platelet-derived growth factor (PDGF) beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants were made in which Tyr579, Tyr581 or both were replaced with phenylalanine residues; the receptor mutants were stably expressed in porcine aortic endothelial cells. Compared with the wild-type receptor, the Y579F and Y581F mutants were less able to mediate association with and activation of the Src family tyrosine kinases. The ability of these phosphorylation sites to mediate directly the binding of the Src family proteins was also demonstrated by using phosphotyrosine-containing synthetic peptides representing the juxtamembrane sequence of the receptor. Both the Y579F and Y581F mutants were similar to the wild-type receptor with regard to their protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. A conclusive evaluation of the role of the Src family members in signal transduction could, however, not be made since our attempt to prevent completely the association by mutation of both Tyr579 and Tyr581, resulted in loss of kinase activity and was therefore not informative. The present data, together with previous observations, demonstrate a high degree of specificity in the interaction between different autophosphorylation sites in the PDGF beta-receptor and downstream components in the signal transduction pathway.     

Regulation of proliferation and differentiation of myoblasts derived from adult mouse skeletal muscle by specific isoforms of PDGF

The expression of receptors and the mitogenic response to PDGF by C2 myoblasts, derived from adult mouse skeletal muscle, was investigated. Employing 125I-PDGF binding assays, we showed that the cells exhibit high level binding of PDGF-BB (approximately 165 x 10(3) molecules/cell at saturation) and much lower binding of the PDGF-AA and PDGF-AB (6-12 x 10(3) molecules/cell at saturation). This indicates that the C2 myoblasts express high levels of PDGF receptor beta-subunits and low levels of alpha-subunits. PDGF-BB enhances the proliferation of C2 cells maintained in 2% FCS by about fivefold. PDGF-AB had a moderate effect on cell proliferation (less than twofold) and PDGF-AA had no effect. Inverse effects of PDGF isoforms on the frequency of differentiated myoblasts were observed; the frequency of myosin-positive cells was reduced in the presence of PDGF-BB while PDGF-AA and PDGF-AB had no effect. PDGF may thus act to increase the number of myoblasts that participate in muscle regeneration following muscle trauma by stimulating the proliferation and by inhibiting the differentiation of myogenic cells.     

Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.

Sphingosine-1-phosphate, a sphingolipid metabolite, is involved in the mitogenic response of platelet-derived growth factor (PDGF) and is formed by activation of sphingosine kinase. We examined the effect of PDGF on sphingosine kinase activation in TRMP cells expressing wild-type or various mutant betaPDGF receptors. Sphingosine kinase was stimulated by PDGF in cells expressing wild-type receptors but not in cells expressing kinase-inactive receptors (R634). Cells expressing mutated PDGF receptors with phenylalanine substitutions at five major tyrosine phosphorylation sites 740/751/771/1009/1021 (F5 mutants), which are unable to associate with PLCgamma, phosphatidylinositol 3-kinase, Ras GTPase-activating protein, or protein tyrosine phosphatase SHP-2, not only failed to increase DNA synthesis in response to PDGF but also did not activate sphingosine kinase. Moreover, mutation of tyrosine-1021 of the PDGF receptor to phenylalanine, which impairs its association with PLCgamma, abrogated PDGF-induced activation of sphingosine kinase. In contrast, PDGF was still able to stimulate sphingosine kinase in cells expressing the PDGF receptor mutated at tyrosines 740/751 and 1009, responsible for binding of phosphatidylinositol 3-kinase and SHP-2, respectively. In agreement, PDGF did not stimulate sphingosine kinase activity in F5 receptor 'add-back' mutants in which association with the Ras GTPase-activating protein, phosphatidylinositol 3-kinase, or SHP-2 was individually restored. However, a mutant PDGF receptor that was able to bind PLCgamma (tyrosine-1021), but not other signaling proteins, restored sphingosine kinase sensitivity to PDGF. These data indicate that the tyrosine residue responsible for binding of PLCgamma is required for PDGF-induced activation of sphingosine kinase. Moreover, calcium mobilization downstream of PLCgamma, but not protein kinase C activation, appears to be required for stimulation of sphingosine kinase by PDGF.-Olivera, A., Edsall, J., Poulton, S., Kazlauskas, A., Spiegel, S. Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.     

Identification of two C-terminal autophosphorylation sites in the PDGF beta-receptor: involvement in the interaction with phospholipase C-gamma.

Two novel sites of autophosphorylation were localized to the C-terminal tail of the PDGF beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants in which Tyr1009, Tyr1021 or both were replaced with phenylalanine residues, were expressed in porcine aortic endothelial (PAE) cells. These mutants were similar to the wild type receptor with regard to protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. However, both the Y1009F and Y1021F mutants showed a decreased ability to mediate association with and the tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) compared to the wild type PDGF beta-receptor; in the case of the Y1009F/Y1021F double mutant, no association or phosphorylation of PLC-gamma could be detected. These data show that tyrosine phosphorylation of PLC-gamma is dependent on autophosphorylation of the PDGF beta-receptor at Tyr1009 and Tyr1021.     

Characterization of a Novel Receptor in Toad Retina with Dual Specificity for Insulin and Insulin-Like Growth Factor I

The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin-like growth factor I (IGF-I). Affinity labeling of toad retinal membrane proteins with 125I-insulin resulted in the specific labeling of insulin receptor alpha-subunits of approximately 105 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially reduced (alpha beta-heterodimer) receptors affinity-labeled with 125I-insulin indicated the presence of a disulfide-linked beta-subunit of approximately 95 kDa. Endoglycosidase F digestion of the affinity-labeled alpha-subunits increased their mobility by reducing their apparent mass to approximately 83 kDa. This receptor was not detected by immunoblot analysis with a site-specific antipeptide antibody directed against residues 657-670 of the carboxy terminal of the human insulin receptor alpha-subunit, whereas this antibody did label insulin receptor alpha-subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor beta-subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose alpha-subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF-I binding activities associated with toad retinal membranes.     

Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex.

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.     

Binding of different dimeric forms of PDGF to human fibroblasts: evidence for two separate receptor types

The binding of the three dimeric forms of platelet-derived growth factor (PDGF), PDGF-AA, PDGF-AB and PDGF-BB, to human fibroblasts was studied. Cross-competition experiments revealed the existence of two different PDGF receptor classes: the type A PDGF receptor bound all three dimeric forms of PDGF, whereas the type B PDGF receptor bound PDGF-BB with high affinity and PDGF-AB with lower affinity, but not PDGF-AA. The sizes of the two receptors were estimated with affinity labeling techniques; the A type receptor appeared as a major component of 125 kd and a minor of 160 kd, and the B type receptor as two components of 160 and 175 kd. A previously established PDGF receptor monoclonal antibody, PDGFR-B2, was shown to react with the B type receptor only. The different abilities of the three dimeric forms of PDGF to stimulate incorporation of [3H]TdR into human fibroblasts indicated that the major mitogenic effect of PDGF is mediated via the B type receptor.     

Differential effects of PDGF and PDGF-BB on vascular smooth muscle cells

Several biological effects of recombinant PDGF-BB and PDGF obtained from human platelets were examined with vascular smooth muscle cells. Although PDGF and PDGF-BB were equally potent mitogens for these cells, 5 fold higher levels of PDGF were required to displace 125I-PDGF-BB binding than PDGF-BB itself. Higher concentrations of PDGF relative to PDGF-BB were also required to stimulate the phosphorylation of a 163K protein in membrane preparations. PDGF-BB, but not PDGF, treatment of intact cells resulted in the phosphorylation on tyrosine residues of 168, 53, 48, and 45K proteins. The data suggest that PDGF and PDGF-BB stimulate smooth muscle cell mitogenesis by different mechanisms.     

TNF-alpha suppresses the PDGF beta-receptor kinase

PDGF and TNF-alpha are both known to play important roles in inflammation, albeit frequently by opposing actions. Typically, TNF-alpha can attenuate PDGF beta-receptor signaling. Pretreatment of mouse 3T3 L1 fibroblasts with TNF-alpha greatly diminished their proliferative response to PDGF. However, TNF-alpha affected neither the binding of PDGF-BB to cell surface receptors nor the total amount of PDGF beta-receptor in the cells, but decreased the PDGF-induced in vitro kinase activity of the receptor. The phosphatase inhibitor ortho-vanadate did not prevent this effect. Ortho-phosphate labeling of cells prior to TNF-alpha treatment and PDGF-BB stimulation confirmed a decrease of in vivo phosphorylation of the PDGF beta-receptor. Two-dimensional mapping after tryptic cleavage as well as phosphoamino acid analysis demonstrated a general decrease in phosphorylation of all known tyrosine residues in the PDGF beta-receptor. The exact mechanism for this suppression remains to be clarified.     

Phosphorylation sites in the PDGF receptor with different specificities for binding GAP and PI3 kinase in vivo.

Tyrosine residues have been identified in the human platelet-derived growth factor (PDGF) receptor beta-subunit whose phosphorylation is stimulated by PDGF. These sites are also in vitro autophosphorylation sites. There are a total of three phosphorylation sites in the kinase insert region, tyrosines 740, 751 and 771. Mutagenesis studies show that Tyr740 and 751 are involved in the PDGF-stimulated binding of phosphatidylinositol (PI) 3 kinase, and Tyr771 is required for efficient binding of GAP, the GTPase activator of Ras. The requirement for Tyr751 is only detected at low PDGF receptor levels, suggesting that it increases the affinity of binding of PI3 kinase but is not absolutely required. Small deletions in the kinase insert only 10 residues from Tyr740 and Tyr771 do not significantly reduce binding of PI3 kinase or GAP, indicating that distant sequences are probably unimportant for recognition. The data suggest that the receptor signals to different pathways via different phosphorylated tyrosines, and that certain proteins, such as PI3 kinase, can recognize two phosphorylated tyrosines in a single receptor.     

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.     

Dimerization of B-type platelet-derived growth factor receptors occurs after ligand binding and is closely associated with receptor kinase activation

Platelet-derived growth factor (PDGF) was found to induce dimerization of purified B-type PDGF receptors, as analyzed by sodium dodecyl sulfate gel electrophoresis after covalent cross-linking using disuccinimidyl suberate. PDGF-BB was 20-fold more effective than PDGF-AB; PDGF-AA was without effect. The dimerization was dose-dependent and was maximal at 0.5-2 micrograms/ml PDGF-BB; at higher concentrations dimerization was less abundant. This indicates that dimerization occurred when one PDGF-BB molecule bound two receptor molecules. The dimerization correlated to activation of the tyrosine kinase of the receptor, determined as autophosphorylation, but was not dependent on phosphorylation reactions because it occurred also in the absence of ATP. Furthermore, dimerization of the receptor correlated with the ability to phosphorylate phosphofructokinase, an exogenous substrate. The complex of ligand and receptor dimer was stable; it resisted electrophoresis under nondenaturing conditions, as well as gel chromatography. The present data indicate that intermolecular mechanisms are involved in signal transduction from the external ligand binding domain to the internal effector domains of the B-type PDGF receptor.     

Mechanisms of inhibition by heparin of PDGF stimulated MAP kinase activation in vascular smooth muscle cells

Heparin and heparan are potent inhibitors of vascular smooth muscle cell (VSMC) proliferation. To investigate the mechanisms by which heparin suppresses growth factor stimulated mitogenesis, the present experiments investigated the effects of heparin on platelet-derived growth factor (PDGF) stimulated signal transduction pathways. Heparin treatment substantially inhibited PDGF-BB stimulated rat VSMC growth. Western analysis showed a 30 min PDGF-BB treatment of VSMC induced the tyrosine phosphorylation of multiple protein bands; cotreatment with heparin inhibited mitogen-activated protein (MAP) kinase tyrosine phosphorylation but had little effect on PDGF receptor tyrosine phosphorylation. In-gel kinase assays demonstrated that heparin inhibited PDGF-BB stimulated MAP kinase activity at late (25 min) but not early (10 min) time points. These data indicate that heparin does not inhibit the initial signalling events after PDGF-BB binding but instead acts through an alternate mechanism to inhibit MAP kinase. To investigate if heparin directly stimulates tyrosine phosphatase-mediated suppression of MAP kinase, we treated VSMC with orthovanadate, a tyrosine phosphatase inhibitor. Heparin inhibited MAP kinase tyrosine phosphorylation after orthovanadate treatment, indicating that heparin does not suppress MAP kinase by enlistment of a tyrosine phosphatase. Experiments were performed to investigate signalling pathways upstream of MAP kinase. To determine if protein kinase C (PKC) mediates PDGF-BB, serum, and EGF stimulation of MAP kinase, we treated VSMC overnight with phorbol ester (PMA) to downregulate PKC. Abolition of conventional and novel PKC activity significantly suppressed both serum and PDGF-BB induced MAP kinase activation, indicating protein kinase C is an important mediator for these mitogens. In contrast, downregulation of these PKC isoforms had little effect on EGF stimulation of MAP kinase. As heparin inhibits PDGF and serum but not EGF stimulation of MAP kinase, these data precisely correlate heparin inhibition of MAP kinase with activation through PKC-dependent pathways. Immunoprecipitation analysis found that heparin inhibited serum, PMA, and PDGF but not EGF induced raf-1 phosphorylation. These studies demonstrate that heparin did not block PDGF-BB receptor activation, which initiates the mitogenic signalling cascade. Heparin did inhibit specific postreceptor second messenger signals, such as the late phase activation of MAP kinase, which may be mediated by suppression of PKC-dependent pathways. J. Cell. Physiol. 172:69–78, 1997. © 1997 Wiley-Liss, Inc.     

Differential regulation of expression of two platelet-derived growth factor receptor subunits by transforming growth factor-beta

The binding of three radiolabeled isoforms of platelet-derived growth factor (PDGF), 125I-PDGF-AA, 125I-PDGF-AB, and 125I-PDGF-BB, is differentially affected by exposure of quiescent 3T3 cells to transforming growth factor-beta (TGF-beta). By 24 h after exposure to TGF-beta, binding of 125I-PDGF-AA and 125I-PDGF-AB is almost completely lost, whereas binding of 125I-PDGF-BB is reduced by only 40%. The loss of PDGF-binding sites caused by TGF-beta is time- and concentration-dependent and reflects a change in the pattern of expression of receptor subunits; the number of alpha-subunits decreases, and the number of beta-subunits increases. The loss of binding sites for PDGF-AA is accompanied by a decreased mitogenic response to PDGF-AA but not to PDGF-AB or PDGF-BB. These results suggest that TGF-beta may differentially regulate the expression of PDGF-binding sites and the mitogenic responsiveness toward the three PDGF isoforms. TGF-beta did not stimulate synthesis of PDGF A-chain mRNA or PDGF-AA protein, and PDGF-AA receptors could not be restored by the presence of suramin, suggesting that the loss of binding sites may result from direct effects on receptor expression rather than autocrine down-regulation by PDGF-AA.     

Effect of receptor kinase inactivation on the rate of internalization and degradation of PDGF and the PDGF beta-receptor

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.     

Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction.

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.