Differential binding, biological and biochemical actions of recombinant PDGF AA, AB, and BB molecules on connective tissue cells.

We have compared the biological and biochemical properties of recombinant PDGF AA, AB, and BB using three types of fibroblastic cells: NIH/3T3, human skin fibroblast, and fetal bovine aortic smooth muscle. PDGF binding, receptor autophosphorylation, phosphatidyl inositol hydrolysis, as well as chemotactic and mitogenic responses of the cells were analyzed. PDGF-AB and PDGF-BB showed similar receptor binding, receptor autophosphorylation, and potent biological activity for all three of the cell types tested. In contrast, PDGF-AA was biologically active only for the NIH/3T3 cells in which binding sites for PDGF-AA were abundant, but was inactive for bovine aortic smooth muscle cells and human skin fibroblasts in which binding sites for PDGF-AA were absent. PDGF-AA could not induce any biochemical changes in the human skin fibroblasts or smooth muscle cells. Western blot studies with anti-Type alpha and beta PDGF receptor antibodies indicate that the NIH/3T3 cells contained both PDGF alpha and beta receptors, whereas the human skin fibroblasts and bovine smooth muscle cells contained only detectable levels of beta receptors. These results indicate that cells possessing high levels of PDGF beta receptors only are capable of responding equally well to either PDGF AB or BB.     

Ligand activation causes a phosphorylation-dependent change in platelet-derived growth factor receptor conformation

The effect of ligand binding on platelet-derived growth factor (PDGF) receptor conformation was examined using peptide antibodies directed against specific receptor domains. Antiserum 83, which was directed to the receptor's carboxyl terminus (residues 934-951), preferentially immunoprecipitated the ligand-activated form of the PDGF receptor from 35S-labeled BALB/c 3T3 cells. By contrast, two antisera directed against other receptor sequences precipitated unactivated and activated receptors equally well. Denatured receptors were recognized equally by all antisera, even 83. Thus, ligand activation caused a change in PDGF receptor conformation that enhanced accessibility of the antibody to the carboxyl terminus. The activated receptor conformation was induced by three different forms of PDGF (AA and BB homodimers and AB heterodimers) and was reversed by suramin, a polyanionic compound that dissociates PDGF from the receptor. The inhibitory effect of suramin on receptor conformation was abolished by the phosphatase inhibitor, sodium orthovanadate, suggesting that receptor phosphorylation mediated the conformational change. In a cell-free assay, the change in receptor conformation was induced by PDGF only in the presence of ATP and was inhibited by adenyl-5'-yl imidodiphosphate, a nonhydrolyzable analog of ATP. The functional significance of receptor conformation was examined in Chinese hamster ovary fibroblasts transfected with wild-type or mutated forms of the PDGF receptor. When receptor tyrosine kinase activity was abolished by a mutation of the ATP binding site the receptor no longer underwent PDGF-induced conformational change and did not mediate PDGF-induced mitogenesis even though 125I-PDGF binding was normal. These findings show that ligand binding elicits a phosphorylation-dependent change in PDGF receptor conformation that may be important for receptor function.     

Immunochemical localization of the epitope for a monoclonal antibody that neutralizes human platelet-derived growth factor mitogenic activity.

A monoclonal antibody (mAb), sis 1, generated against human c-sis-encoded platelet-derived growth factor (PDGF) BB, was shown by enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis to recognize human PDGF BB and human platelet PDGF AB but not the human PDGF AA. This monoclonal antibody potently inhibited PDGF receptor-binding and mitogenic activities of both human PDGF BB and PDGF AB but had no effect on PDGF AA. Finally, we demonstrated that an immunoaffinity-purified anti-c-sis peptide antibody (anti-V4) which also blocked binding of PDGF BB to its cognate receptor and competed with mAb sis 1 for binding to PDGF BB. All of these results suggest that mAb sis 1 recognizes an epitope of the c-sis gene product, PDGF BB, that spatially overlaps the V4 surface domain of PDGF BB, immunochemically localizing a region of PDGF BB critical for PDGF receptor binding and activation.     

Actions of platelet-derived growth factor isoforms in mesangial cells

Platelet-derived growth factor (PDGF) occurs as homodimers or heterodimers of related polypeptide chains PDGF-BB, -AA, and -AB. There are two receptors that bind PDGF, termed alpha and beta. The beta receptor recognizes PDGF B chain and is dimerized in response to PDGF BB. The alpha receptor recognizes PDGF B as well as A chains and can be dimerized by the three dimeric forms of PDGF AA, AB, and BB. To characterize PDGF receptor signaling mechanisms and biologic activities in human mesangial cells (MC), we explored the effects of the three PDGF isoforms on DNA synthesis, phospholipase C activation, and PDGF protooncogene induction. PDGF-BB homodimer and AB heterodimer induced a marked increase in DNA synthesis, activation of phsopholipase C, and autoinduction of PDGF A and B chain mRNAs, whereas PDGF-AA homodimer was without effect. The lack of response to PDGF AA could be accounted for by down regulation of the PDGF-alpha receptor since preincubation of MC with suramin restored PDGF AA-induced DNA synthesis. Ligand binding studies demonstrate specific binding of labeled PDGF BB and AB and to a lower extent PDGF AA isoforms to mesangial cells. These results are consistent with predominant expression of PDGF beta receptor in MC, which is linked to phospholipase-C activation. The potent biologic effects of PDGF-AB heterodimer in cells that express very few alpha receptors and do not respond to PDGF AA are somewhat inconsistent with the currently accepted model of PDGF receptor interaction and suggest the presence of additional mechanisms for PDGF isoform binding and activation. © 1994 Wiley-Liss, Inc.     

Effect of the different dimeric forms of the platelet-derived growth factor on cellular responses in mouse Swiss 3T3 fibroblasts

PDGF consists of two polypeptide chains, A and B, and all three possible dimers have been isolated from different sources. Human PDGF, essentially AB, porcine PDGF (BB) and recombinant PDGF-AA were tested on Swiss 3T3 fibroblasts for their ability to stimulate mitogenesis, phosphoinositide turnover and tyrosine phosphorylation of the PDGF receptor. When used in saturating amounts, the three isoforms were equally active in inducing mitogenesis. However, PDGF-AA was less active than AB and BB to induce the phosphorylation of the receptor and the turnover of phosphoinositides (30% and 50%, respectively). These findings suggest that, in Swiss 3T3 fibroblasts, PDGF receptors of the alpha-type are present in a slightly lower amount than beta-type. In addition, the two types of receptor appear to have similar physiological functions.     

Dissociation of the chemotactic and mitogenic activities of platelet- derived growth factor by human neutrophil elastase

Because platelet-derived growth factor (PDGF) may be released at sites where neutrophil proteinases may also be released, we examined the effects of neutrophil elastase and cathepsin G upon the chemotactic and mitogenic activities of PDGF. Elastase abolished the chemotactic activity of PDGF for fibroblasts but had no effect on its chemotactic activity for monocytes, or on its mitogenic activity for 3T3 cells or its capacity to bind to 3T3 cells. Cathepsin G had no effect upon the chemotactic or mitogenic activities of PDGF. In contrast, trypsin eliminated the chemotactic activity of PDGF for monocytes and fibroblasts and the mitogenic activity of PDGF. After reduction and alkylation, PDGF retained full chemotactic activity for fibroblasts and monocytes but exhibited no mitogenic activity and only limited binding to 3T3 cells. These results indicate separate domains on PDGF for fibroblast chemotactic and mitogenic activity and for monocyte and fibroblast chemotactic activity and raise the possibility that the biological activities of PDGF may be modified selectively in vivo. The findings further suggest that the majority of PDGF receptors on fibroblasts mediate mitogenic activity and that only a minority of the PDGF receptors on fibroblasts are responsible for chemotactic activity.     

Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro

Phospholipase C-gamma (PLC-gamma) was rapidly phosphorylated on tyrosines and serines following PDGF and EGF treatment of quiescent 3T3 mouse fibroblasts and A431 human epidermoid cells, respectively, PDGF treatment increased PLC-gamma phosphorylation within 30 sec. This lasted for up to 1 hr, and occurred at high stoichiometry. Continuous receptor occupancy was required to maintain this phosphorylation. Three major sites of tyrosine phosphorylation were detected in PLC-gamma, two of which were phosphorylated in EGF-treated A431 cells. Under certain conditions PDGF receptor coimmunoprecipitated with PLC-gamma, suggesting that PDGF receptor can phosphorylate PLC-gamma directly. Indeed, purified PDGF or EGF receptor phosphorylated purified PLC-gamma on tyrosines identical to those phosphorylated in vivo. Tyrosine phosphorylation of PLC-gamma was not induced by bombesin, TPA, or insulin. Stimulation of PLC-gamma tyrosine phosphorylation and the reported ability of PDGF and EGF to induce phosphatidylinositol turnover in different cells were strongly correlated. We propose that tyrosine phosphorylation of PLC-gamma by PDGF and EGF receptors leads to its activation, and a consequent increase in phosphatidylinositol turnover.     

Ligand-induced interaction between alpha- and beta-type platelet-derived growth factor (PDGF) receptors: role of receptor heterodimers in kinase activation.

Two types of PDGF receptors have been cloned and sequenced. Both receptors are transmembrane glycoproteins with a ligand-stimulatable tyrosine kinase site. We have shown earlier that ligand-induced activation of the beta-type PDGF receptor is due to the conversion of the monomeric form of the receptor to the dimeric form [Bishayee et al. (1989) J. Biol. Chem. 264, 11699-11705]. In the present studies, we have established the ligand-binding specificity of two receptor types and extended it further to investigate the ligand-induced association state of the alpha-receptor and the role of alpha-receptor in the activation of beta-receptor. These studies were conducted with cells that express one or the other type of PDGF receptor as well as with cells that express both types of receptors. Moreover, ligand-binding characteristics of the receptor were confirmed by immunoprecipitation of the receptor-125I-PDGF covalent complex with type-specific anti-PDGF receptor antibodies. These studies revealed that all three isoforms of PDGF bind to alpha-receptor, and such binding leads to dimerization as well as activation of the receptor. In contrast, beta-receptor can be activated only by PDGF BB and not by PDGF AB or PDGF AA. However, by using antipeptide antibodies that are specific for alpha- or beta-type PDGF receptor, we demonstrated that in the presence of alpha-receptor, beta-receptor kinase can be activated by PDGF AB. We present here direct evidence that strongly suggests that such PDGF AB induced activation of beta-receptor is due to the formation of a noncovalently linked alpha-beta receptor heterodimer.     

A comparison of the platelet-derived growth factor-dependent tyrosine kinase activity in sparse and confluent fibroblasts

Confluent (density-inhibited) human foreskin fibroblasts require a higher concentration of platelet-derived growth factor (PDGF) to elicit a mitogenic response than do sparse (nondensity-inhibited) fibroblasts. The PDGF receptor number and apparent affinity were similar in the two preparations of cells. The intrinsic kinase activity of the PDGF receptor from sparse and confluent fibroblasts was therefore examined in an attempt to explain the differential mitogenic response to PDGF. When membranes from sparse and confluent cells containing equal PDGF binding capacity were incubated with increasing concentrations of PDGF, the putative PDGF receptor (a 180-kD component), was phosphorylated on its tyrosyl residues to a similar extent. The time course of tyrosine phosphorylation of the PDGF receptor from sparse and confluent cell membranes was also found to be similar. To determine whether the phosphorylation of the PDGF receptor from isolated membranes differed from the analogous phosphorylation in intact cells, sparse and confluent fibroblasts were metabolically labeled with [32P]H3PO4, stimulated with PDGF, solubilized, and the cell proteins were immunoprecipitated with a phosphotyrosine-specific antibody. The extent of PDGF-dependent tyrosine phosphorylation of the PDGF receptor from sparse vs. confluent fibroblasts was quite similar. The time course of the tyrosine dephosphorylation of the PDGF receptor was also similar in the two populations. Because comparable extents of PDGF-induced tyrosine phosphorylation of the PDGF receptor were observed despite the differential PDGF-induced mitogenic response of sparse and confluent fibroblasts, we tentatively conclude that 1) PDGF-dependent tyrosine phosphorylation of the PDGF receptor is not tightly coupled to the propagation of the mitogenic signal and 2) density-dependent inhibition of growth does not reflect any measurable change in the quantity of kinase activity of the PDGF receptor.     

Ligand-independent activation of the platelet-derived growth factor beta receptor: requirements for bovine papillomavirus E5-induced mitogenic signaling.

The E5 protein of bovine papillomavirus type 1 binds to and activates the endogenous platelet-derived growth factor (PDGF) beta receptor in fibroblasts, resulting in cell transformation. We have developed a functional assay to test the ability of PDGF beta receptor mutants to mediate a mitogenic signal initiated by the E5 protein. Lymphoid Ba/F3 cells are strictly dependent on interleukin-3 for growth, but coexpression of the wild-type PDGF beta receptor and the E5 or v-sis-encoded protein generated a mitogenic signal which allowed Ba/F3-derived cells to proliferate in the absence of interleukin-3. In these cells, the E5 protein bound to and caused increased tyrosine phosphorylation of both the mature and the precursor forms of the wild-type PDGF beta receptor. The tyrosine kinase activity of the receptor was necessary for E5-induced receptor tyrosine phosphorylation and mitogenic activity but not for complex formation with the E5 protein. In contrast, the PDGF-binding domain of the receptor was not required for complex formation with the E5 protein, E5-induced tyrosine phosphorylation or mitogenic activity, demonstrating that E5-mediated receptor activation is ligand independent. Analysis of receptor mutants lacking various combinations of tyrosine phosphorylation sites revealed that the E5 and v-sis-encoded proteins display similar requirements for signaling and suggested that the wild-type PDGF beta receptor can generate multiple independent mitogenic signals. Importantly, these mutants dissociated two activities of the PDGF beta receptor tyrosine kinase, both of which are required for sustained mitogenic signaling: (i) receptor autophosphorylation and creation of binding sites for SH2 domain-containing proteins and (ii) phosphorylation of substrates other than the receptor itself.     

Differential regulation of the expression of proteinases/antiproteinases in fibroblasts. Effects of interleukin-1 and platelet-derived growth factor.

We have previously reported that polypeptide growth factors had an anti-inflammatory effect by decreasing the cytokine-enhanced expression of factor B (FB), an activator of the alternative complement pathway, in human fibroblasts. To further characterize the role of cytokines and growth factors in the inflammatory/repair continuum, we have studied the effects of interleukin-1 (IL-1) and platelet-derived growth factor (PDGF) on the expression of metalloproteinases/antiproteinases of the extracellular matrix in cultured human fibroblasts. Co-incubation of IL-1 and PDGF synergistically increased the expression of stromelysin and interstitial collagenase to 23-fold (for both proteins) over background, while PDGF decreased the IL-1-enhanced expression of FB by 82%. PDGF, but not IL-1, alone or in combination, increased the synthesis of tissue inhibitor of metalloproteinases. RNA blot analysis indicated that the changes in protein synthesis were regulated at a pretranslational level. Cycloheximide treatment indicated that the effects of PDGF on the metalloproteinases/antiproteinases were not protein-dependent, in contrast to results obtained for FB. The effect of the three dimeric forms of PDGF (AA, AB, and BB) on the synthesis of metalloproteinases and FB was also analyzed. The effects were qualitatively similar for each of the dimeric forms; however, the BB and AB isoforms had considerably greater effects than PDGF-AA. It has been reported that the PDGF receptors found in human fibroblasts have higher binding affinity for the BB and AB isoforms of the growth factor. The results presented in this paper are in accord with the possibility that differences in the biological activity of the three isoforms of PDGF are due to differences in the number or affinity of the binding sites of the target cells, rather than to different activation pathways of the receptor. Thus, PDGF increased cytokine effects on metalloproteinases, while decreasing cytokine effects or complement activator FB. The net effect of these changes may be to decrease inflammation and enhance remodeling early in repair and to enhance matrix stability later in the repair process.     

Phospholipase C-gamma, a substrate for PDGF receptor kinase, is not phosphorylated on tyrosine during the mitogenic response to CSF-1.

Quiescent mouse NIH3T3 cells expressing a transduced human c-fms gene encoding the receptor for colony stimulating factor-1 (CSF-1) were stimulated with mitogenic concentrations of platelet-derived growth factor (PDGF) or CSF-1. Immunoprecipitated phospholipase C-gamma (PLC-gamma) was phosphorylated on tyrosine and calcium was mobilized following treatment of intact cells with PDGF. In contrast, only trace amounts of phosphotyrosine were incorporated into PLC-gamma and no intracellular calcium signal was detected after CSF-1 stimulation. Similarly, CSF-1 treatment did not stimulate phosphorylation of PLC-gamma on tyrosine in a CSF-1-dependent. SV40-immortalized mouse macrophage cell line that expresses high levels of the CSF-1 receptor. In fibroblasts, antiserum to PLC-gamma co-precipitated a fraction of the tyrosine phosphorylated form of the PDGF receptor (PDGF-R) after ligand stimulation, implying that phosphorylated PDGF-R and PLC-gamma were associated in a stable complex. Pre-treatment of cells with orthovanadate also led to tyrosine phosphorylation of PLC-gamma which was significantly enhanced by PDGF, but not by CSF-1. Thus, although the PDGF and CSF-1 receptors are structurally related and appear to be derived from a single ancestor gene, only PDGF-induced mitogenesis in fibroblasts correlated with tyrosine phosphorylation of PLC-gamma.     

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.     

Characterization of baculovirus-expressed human alpha and beta platelet-derived growth factor receptors.

In an effort to biochemically characterize PDGF receptors and their mechanism of activation, recombinant baculovirus vectors containing the cDNAs of the human alpha PDGF receptor or beta PDGF receptor were engineered. Characterization of recombinant PDGF receptor expression in infected Sf9 insect cells by immunoblot analysis with specific PDGF receptor peptide antisera revealed that the alpha and beta PDGF receptor gene products were translated as 160- and 165-kDa transmembrane proteins, respectively. Ligand binding analysis demonstrated saturable, high-affinity binding of either 125I-labeled PDGF AA or 125I-labeled PDGF BB to Sf9 cells expressing the recombinant alpha PDGF receptor. In contrast, recombinant beta PDGF receptor expressing Sf9 cells showed high-affinity binding only for PDGF BB. Analysis of the kinetics of PDGF receptor expression demonstrated that receptor number increased dramatically from 24- to 48-h postinfection. Early in infection, the PDGF receptors were present in low numbers, lacked tyrosine phosphorylation, and exhibited ligand-dependent tyrosine phosphorylation. However, with increasing time postinfection and increasing receptor number, the PDGF receptors became constitutively tyrosine-phosphorylated in serum-free culture medium. Cross-linking studies revealed that receptor activation involved ligand-independent receptor dimer formation at high receptor number. Thus, these results strongly suggest that PDGF stabilizes and increases the frequency of PDGF receptor interaction, which ultimately results in PDGF receptor activation and intracellular signaling.     

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.     

PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex

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.     

Identification of c-erbB-3 binding sites for phosphatidylinositol 3''-kinase and SHC using an EGF receptor/c-erbB-3 chimera.

c-erbB-3 is a member of the type I (EGF receptor-related) family of growth factor receptors for which no ligand has been identified. To facilitate ligand stimulation we have constructed a chimeric receptor which possesses an activatable kinase and promotes the growth of NIH 3T3 fibroblasts. In this study we have shown that SHC and phosphatidylinositol 3'-kinase bind to the activated EGF receptor/c-erbB-3 chimera. Whereas p85 is not phosphorylated to a significant extent, SHC appears to be a major substrate for phosphorylation on tyrosine. In contrast to EGF receptor and c-erbB-2, we were unable to detect binding of activated c-erbB-3 to GRB2. Using synthetic peptides corresponding to each of 13 potential phosphorylation sites on c-erbB-3, we have shown that tyrosine 1309 is responsible for SHC binding. Peptides containing the motif YXXM inhibit p85 association. By comparison with recently reported SHC binding sites on Middle T antigen and Trk we have identified a SHC binding motif, NPXY.     

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.     

Effect of the growth conditions on the expression of cell-surface-associated platelet-derived growth factor receptors in mouse fibroblasts

The conditions affecting the appearance and disappearance of platelet-derived growth factor (PDGF) receptors from the pool of active cell surface-associated receptors were studied. Receptor molecules were revealed in intact Swiss 3T3 fibroblasts by their ability to bind 125I-labeled PDGF and, due to their property to become phosphorylated in tyrosine following ligand binding, by antibodies to phosphotyrosine. PDGF receptor molecules were found to be quite scarce in exponentially growing fibroblasts as compared to quiescent cells. When growing cells were either shifted to a medium containing plasma or received suramin in the culture medium, cell surface-associated PDGF receptors largely increased. This process required about 12 h. Incubation of quiescent cells in serum, but not in plasma, induced a slow decrement of ligand-activatable receptors. In the presence of PDGF the rate of receptor removal from the cell surface was very rapid and was a function of the PDGF concentration. Quiescent cells deprived of cell-surface receptors by incubation with PDGF reexpressed PDGF receptors in about 14 h.     

Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor.

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.