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.     

Evidence for the platelet-derived growth factor-stimulated tyrosine phosphorylation of the platelet-derived growth factor receptor in vivo. Immunopurification using a monoclonal antibody to phosphotyrosine

The addition of platelet-derived growth factor (PDGF) to intact BALB/c 3T3 cells results in the rapid (less than 1 min), dose-dependent phosphorylation of a number of proteins that could be isolated by a monoclonal antiphosphotyrosine antibody. The predominant tyrosinephosphorylated protein shared many characteristics with the PDGF receptor, including its molecular weight (170,000), isoelectric point (pI of about 4.2), its binding to DEAE-cellulose, and its pattern of binding to lectins. This 170-kDa protein, labeled with [35S] methionine, was substantially purified from PDGF-stimulated cells using the monoclonal anti-phosphotyrosine antibody but was not significantly immunopurified from unstimulated cells. At 37 degrees C, phosphorylation of the 170-kDa protein was maximal by 5-10 min of exposure to PDGF, and thereafter decreased rapidly. However, at 4 degrees C, the phosphorylation continued to increase after 3 h of exposure to PDGF. Subsequently, shifting the cells from 4 to 37 degrees C resulted in an additional rapid burst of tyrosine phosphorylation. Among the other PDGF-stimulated molecules, the most prominent and consistently observed was a cytosolic, acidic (pI of about 4.2), 74-kDa protein. These findings indicate that the action of PDGF in vivo is associated with the rapid and transient tyrosine phosphorylation of several membrane and cytosolic proteins; the most prominent of these proteins, isolated by monoclonal antibody to phosphotyrosine, is likely to be the PDGF receptor. The use of this antibody provides a new approach for purification of the PDGF receptor.     

Purification of a heparin binding FGF receptor (HB-FGFR) from adult bovine brain membranes

A new form of high affinity fibroblast growth factor receptor has been purified from adult bovine brain membranes. Purification was performed by chromatography on DEAE-Trisacryl and wheat germ agglutinin-agarose followed by FGF-2 affinity chromatography. Affinity labeling of purified fractions with 125I-FGF-2 showed after cross-linking a 170-kDa complex, suggesting the existence of a 150-kDa FGF receptor. No cross-reactivity with anti-FGF receptor 1 (FGFR-1 or flg) or with anti-receptor 2 (FGFR-2 or bek) antibodies could be detected with this partially purified receptor. Heparitinase treatment of the partially purified FGF receptor abolished the formation of the ligand receptor complex. The complex was restored in the presence of heparin in a dose dependent fashion, supporting the idea that heparin-like molecules are needed for proper binding. Further purification of the receptor was achieved by heparin-Sepharose affinity chromatography and yielded a purification of over 320,000-fold. The purified receptor fraction was radiolabeled and loaded on RPLC C4 column. Eluted fractions were analysed by SDS-PAGE. A major 150-kDa band was detected. These data show for the first time a new form of FGF receptor isolated from bovine brain membranes. This purified receptor displays affinity for heparin and was therefore named heparin binding FGF receptor (HB-FGFR). It remains unclear whether the receptor is a proteo-heparin sulfate or whether heparans are strongly associated and therefore are copurified. Large scale preparations are in progress for core protein structure studies.     

Differential processing and turnover of the oncogenically activated neu/erb B2 gene product and its normal cellular counterpart

In nontransformed DHFR/G-8 cells (NIH 3T3 cells transfected with normal rat neu gene), the normal neu gene product was initially synthesized as a 170-kDa protein bearing endoglycosidase H-sensitive oligosaccharide chains and was then processed to a 175-kDa mature form with endoglycosidase H-resistant, endoglycosidase F-sensitive oligosaccharide chains. Most of this 175-kDa mature form appeared on the cell surface 2 h following synthesis and showed a half-life of approximately 3 h. In the presence of a growth factor(s) partially purified from bovine kidney, the half-life of this 175-kDa normal neu gene product was shortened to less than 30 min. In B104-1-1 cells (NIH 3T3 cells transfected with neu gene activated oncogenically by a point mutation that changes a valine residue to a glutamic acid residue in the putative transmembrane region), the oncogenically activated neu gene product was also synthesized as a 170-kDa precursor with endoglycosidase H-sensitive oligosaccharide chains. However, this 170-kDa precursor diminished very fast and was only partially processed to a 185-kDa mature form which exhibited a half-life of less than 30 min. The 185-kDa activated neu gene product possessed an unidentified post-translational modification in addition to N-linked oligosaccharide chains. Both the precursor and mature forms of the mutationally activated neu gene product showed increased tyrosine-specific phosphorylation as compared with those of their normal counterparts in DHFR/G-8 cells. The mutationally activated neu gene product in B104-1-1 cells shared several features which have been reported previously for the ligand-activated platelet-derived growth factor receptor in v-sis- or c-sis-transformed cells. These properties include: 1) accelerated turnover of the precursor and mature forms compared with the rates of turnover of its normal counterparts, 2) insensitivity of this rapid turnover to lysosomotropic amines, and 3) increased in vivo tyrosine-specific phosphorylation of both the precursor and mature forms. These findings suggest that the mutationally activated neu gene product may transform the cells by mimicking ligand-induced activation.     

Epidermal growth factor receptor transactivation is regulated by glucose in vascular smooth muscle cells

We hypothesized that glucose-mediated alterations in vascular smooth muscle cell signal transduction contribute to diabetic complications. We found enhanced AngII activation of Akt and extracellular ERK1/2 in vascular smooth muscle cells incubated with high glucose (27.5 mM) compared with low glucose (5.5 mM). Because AngII-mediated transactivation of the epidermal growth factor receptor (EGFR) is important in Akt and ERK1/2 activation, we studied the effects of glucose on EGFR function. The EGFR in cells cultured for 48 h in low glucose was smaller (145 kDa) than the EGFR in cells cultured with high glucose (170 kDa). The shift from the 170-kDa isoform to the 145-kDa isoform was reversible and dependent upon glucose concentration with EC50 approximately 1 mM. N-Glycosylation was responsible because peptide N-glycosidase F treatment of isolated 170-kDa EGFR yielded a single band at 145 kDa. Cell surface biotinylation showed that the 145-kDa EGFR was present on plasma membrane. AngII and other G-protein-coupled receptor ligands known to transactivate EGFR phosphorylated the 170-kDa EGFR but not the 145-kDa EGFR, whereas EGF, heparin-binding EGF-like growth factor, and transforming growth factor-alpha phosphorylated both receptors. Subcellular fractionation showed that the 145-kDa receptor localized to a different plasma membrane domain than the 170-kDa receptor. These results establish a novel mechanism by which glucose-dependent EGFR N-glycosylation modulates AngII signal transduction and suggest a potential mechanism for pathogenic effects of AngII in diabetic vasculopathy.     

Different forms of the epidermal growth factor receptor kinase have different autophosphorylation sites

Limited proteolysis converts the native (Mr 170 000) epidermal growth factor (EGF) receptor to the Mr 150 000 form of the receptor. Calcium-activated, neutral protease (purified to homogeneity from beef lung), chymotrypsin, and elastase were all similarly effective in generating the 150-kilodalton (150-kDa) form of the receptor in detergent-solubilized, membrane vesicles shed from A-431 cells. The rate of autophosphorylation with [gamma-32P]ATP of the 150-kDa form was only 10% of the rate with the native receptor. This decreased rate was not due to loss of kinase activity, since the phosphorylation of angiotensin was virtually unchanged after limited proteolysis of the native receptor kinase. However, maps of elastase-produced peptides from 170-kDa forms and elastase-generated 150-kDa forms of the EGF receptor showed that the major autophosphorylation sites in these two forms were totally different. Confirming this difference in autophosphorylation sites was the finding that the 32P label in the autophosphorylated native receptor could not be recovered in the 150-kDa form following proteolysis. This label was quantitatively recovered in 30-15-kDa peptide fragments generated simultaneously with the 150-kDa form of the receptor. Therefore, the decreased autophosphorylation of the 150-kDa form results from the loss of preferred autophosphorylation sites on the native receptor. Only 1-3% of the phosphate incorporated in the native receptor during autophosphorylation could be found on the 150-kDa autophosphorylation sites. Hence, autophosphorylation of the tyrosine sites in the 150-kDa form of the EGF receptor is markedly enhanced by removing the major sites autophosphorylated on the native form of the receptor.     

Calcium-activated neutral protease purified from beef lung: properties and use in defining structure of epidermal growth factor receptors

Ca2+-Requiring proteases degrade cytosolic and integral membrane proteins as well as alter, by limited proteolysis, the activity of certain protein kinases. When cells are lysed, a Ca2+-requiring protease degrades the epidermal growth factor (EGF) receptor, an integral membrane protein with an intrinsic kinase activity, from its 170-kDa form to a 150-kDa form. This Ca2+-requiring protease has all of the characteristics of calcium-activated neutral protease (CANP). To show that CANP is the protease uniquely responsible for the degradation of the native EGF receptor in vitro, CANP was highly purified from beef lung. This affinity purified CANP had properties previously described for other CANPs: heterodimer of 80 and 30 kDa; neutral pH optimum; activation by millimolar Ca2+; and inhibition by an endogenous, heat-stable proteinaceous inhibitor, by leupeptin, and by sulfhydryl alkylating agents. Using the EGF receptor labeled by covalent attachment to 125I-EGF, this purified CANP quantitatively generated the 150-kDa form from the native receptor in A-431 cell membranes. As with the native receptor, the 150-kDa receptor forms produced by the endogenous Ca2+-requiring protease, by CANP, by chymotrypsin, and by elastase were all capable of EGF-stimulated autophosphorylation. When the 150-kDa receptor forms were generated by the three exogenously added proteases, autophosphorylation with [gamma-32P]ATP followed by trypsinization produced 32P-labeled peptides that were not the same. However, the tryptic 32P-labeled peptides from the autophosphorylated 150-kDa receptor form produced by CANP or by the endogenous Ca2+-requiring protease were identical. These data indicate that CANP is identical to the endogenous Ca2+-requiring protease responsible for producing the autophosphorylating 150-kDa receptor form from the native EGF receptor when cells are lysed.     

Epidermal growth factor receptor in synaptic fractions of the rat central nervous system.

Functional relationships between epidermal growth factor (EGF) and neural tissues have of late attracted increasing interest. However, in spite of reported EGF effects on neurons, the expression of the EGF receptor (EGF-R) has not yet been unambiguously demonstrated in these cells. This 170-kDa protein bears an intracellular tyrosine kinase domain in which activity is ligand-dependent. We give definitive evidence here for its presence in neonatal and adult rat neurons showing also, for the first time, its binding and functional tyrosine kinase activities in the synaptic region. Immunohistochemistry using a polyclonal antibody prepared against the receptor purified from rat liver showed positive staining localized exclusively to neurons without regionalization to any particular brain zone. Binding studies made in Percoll-obtained synaptosomes revealed specific high affinity 125I-EGF binding sites (Kd, 1.42 x 10(-10) +/- 0.58 M) accounting for 17% of total binding and a great majority of low affinity (Kd, 2.55 x 10(-9) +/- 0.35 M) binding sites. Higher binding capacity was found in synaptosomal fractions obtained from newborn rats. The identity of the synaptosomal EGF binding activity with the 170-kDA EGF-R protein was demonstrated by cross-linking experiments. Furthermore, EGF-Affi-Prep affinity chromatography adsorbs a 170-kDa protein with EGF-R immunoreactivity from whole homogenates of adult rat brain. Phosphorylation assays made in freeze-thawed or intact synaptosomes showed EGF-induced tyrosine phosphorylation in the range of 170-, 126-150-, 124-, 113-, 98-, and 70-kDa proteins including the EGF-R. Thus, the EGF-R/EGF regulatory system could have a role in synaptic function that remains to be explored.     

Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases

Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites.     

Tethering of the platelet-derived growth factor beta receptor to G-protein-coupled receptors. A novel platform for integrative signaling by these receptor classes in mammalian cells

Here we provide evidence to show that the platelet-derived growth factor beta receptor is tethered to endogenous G-protein-coupled receptor(s) in human embryonic kidney 293 cells. The tethered receptor complex provides a platform on which receptor tyrosine kinase and G-protein-coupled receptor signals can be integrated to produce more efficient stimulation of the p42/p44 mitogen-activated protein kinase pathway. This was based on several lines of evidence. First, we have shown that pertussis toxin (which uncouples G-protein-coupled receptors from inhibitory G-proteins) reduced the platelet-derived growth factor stimulation of p42/p44 mitogen-activated protein kinase. Second, transfection of cells with inhibitory G-protein alpha subunit increased the activation of p42/p44 mitogen-activated protein kinase by platelet-derived growth factor. Third, platelet-derived growth factor stimulated the tyrosine phosphorylation of the inhibitory G-protein alpha subunit, which was blocked by the platelet-derived growth factor kinase inhibitor, tyrphostin AG 1296. We have also shown that the platelet-derived growth factor beta receptor forms a tethered complex with Myc-tagged endothelial differentiation gene 1 (a G-protein-coupled receptor whose agonist is sphingosine 1-phosphate) in cells co-transfected with these receptors. This facilitates platelet-derived growth factor-stimulated tyrosine phosphorylation of the inhibitory G-protein alpha subunit and increases p42/p44 mitogen-activated protein kinase activation. In addition, we found that G-protein-coupled receptor kinase 2 and beta-arrestin I can associate with the platelet-derived growth factor beta receptor. These proteins play an important role in regulating endocytosis of G-protein-coupled receptor signal complexes, which is required for activation of p42/p44 mitogen-activated protein kinase. Thus, platelet-derived growth factor beta receptor signaling may be initiated by G-protein-coupled receptor kinase 2/beta-arrestin I that has been recruited to the platelet-derived growth factor beta receptor by its tethering to a G-protein-coupled receptor(s). These results provide a model that may account for the co-mitogenic effect of certain G-protein-coupled receptor agonists with platelet-derived growth factor on DNA synthesis.     

Epidermal growth factor stimulates tyrosine phosphorylation of specific proteins in permeabilized human fibroblasts

We have investigated the epidermal growth factor (EGF)-stimulated tyrosine-specific protein kinase activity in quiescent cultures of diploid human fibroblasts that have a well characterized mitogenic response to EGF. We developed a method of permeabilizing cells with digitonin or other agents that permitted the rapid labeling of cellular proteins with exogenously added [gamma-32P]ATP while allowing only about 25% of marker cytosolic enzymes to escape from the cells. When phosphatases were inhibited with zinc and vanadate, EGF induced up to 8-fold stimulation of the incorporation of radioactivity from [gamma-32P]ATP into a 35-kDa band on sodium dodecyl sulfate gels. Alkali treatment of gels showed that EGF stimulated the phosphorylation of bands with apparent molecular masses of 170, 45, 35, 26, 22, and 21 kDa. Phosphoamino acid analysis was performed on the 170- and 35-kDa bands and revealed that the EGF-stimulated phosphorylation was on tyrosyl residues. The 35-kDa band was resolved into four spots by two-dimensional gel electrophoresis. The most acidic form was the most prominent and it was precipitated by an antiserum against a 35-kDa protein from A-431 cells; heretofore, this protein has only been reported to be phosphorylated in an EGF-dependent manner by A-431 membranes in vitro (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). This antiserum also precipitated a 35-kDa phospho-protein from extracts of intact [32P]orthophosphate-labeled fibroblasts which was phosphorylated on tyrosine in an EGF-dependent manner. None of the forms of the 35-kDa phosphoproteins labeled in permeabilized cells were immunologically related to the 34-kDa protein that is a substrate for the tyrosyl kinase encoded by Rous sarcoma virus. Other mitogens (serum, insulin, platelet-derived growth factor, and thrombin) did not detectably stimulate phosphorylation in permeabilized cells.     

Inhibition of tyrosine kinase activity of the epidermal growth factor (EGF) receptor by a truncated receptor form that binds to EGF: role for interreceptor interaction in kinase regulation.

The tyrosine kinase activity of the epidermal growth factor (EGF) receptor is regulated by a truncated receptor of 100 kilodaltons (kDa) that contains the EGF-binding site but not the kinase domain. The inhibition of kinase is not due to competition for available EGF or for the kinase substrate-binding site. Chemical cross-linking studies suggest that the 100-kDa receptor may form a heterodimer with the intact EGF receptor. Structurally related receptor kinases, such as the platelet-derived growth factor receptor, the insulin receptor, and the Neu receptor, were not inhibited by the 100-kDa receptor. The results indicate that (i) the inhibition was specific for the EGF receptor, (ii) the kinase domain had little or no role in determining target specificity, and (iii) the regulation of kinase may be due to a specific interaction of the 100-kDa receptor with the ligand-binding domain of the EGF receptor kinase.     

Characteristics of 30-, 63-, and 89-kilodalton proteins whose secretion from mouse fibroblasts is altered by beta-interferon

Quiescent mouse BALB/c-3T3 cells were treated with beta-interferon to induce the secretion of proteins of 30 and 89 kDa and with a platelet-derived growth factor preparation to induce the secretion of a 63-kDa protein. To label the secreted proteins the cultures were supplemented with [35S]methionine after addition of the inducer. The proteins in the culture fluid were fractionated resulting in a radioactively pure 63-kDa protein and 30- and 89-kDa protein preparations with residual minor radioactive impurities. The secreted 89-kDa protein shared at least one characteristic with some interferon-induced cell-associated enzymes: it bound double-stranded RNA tightly. The 63-kDa protein was undetectable in the culture fluid from resting BALB/c-3T3 cells and was barely or not at all detectable in the culture fluids from growing BALB/c-3T3 and NIH 3T3 cells, respectively. The protein was, however, among the three major constitutively secreted proteins in the case of growing Kirsten murine sarcoma virus-transformed NIH 3T3 cells. Treatment with 1000 units/ml beta-interferon decreased the accumulation of the 63-kDa protein in the culture fluid of quiescent BALB/c-3T3 cells which had been treated with a platelet-derived growth factor preparation by over 80% and that in the culture fluid of Kirsten murine sarcoma virus-transformed NIH 3T3 cells by about 50%. This decrease was not a consequence of an inhibition of cell growth.     

Growth factor-induced tyrosine phosphorylation of Hrs, a novel 115-kilodalton protein with a structurally conserved putative zinc finger domain.

The activation of growth factor receptor tyrosine kinases leads to tyrosine phosphorylation of many intracellular proteins which are thought to play crucial roles in growth factor signaling pathways. We previously showed that tyrosine phosphorylation of a 115-kDa protein is rapidly induced in cells treated with hepatocyte growth factor. To clarify the structure and possible function of the 115-kDa protein (designated Hrs for hepatocyte growth factor-regulated tyrosine kinase substrate), we purified this protein from B16-F1 mouse melanoma cells by anti-phosphotyrosine immunoaffinity chromatography and determined its partial amino acid sequences. On the basis of the amino acid sequences, we molecularly cloned the cDNA for mouse Hrs. The nucleotide sequence of the cDNA revealed that Hrs is a novel 775-amino-acid protein with a putative zinc finger domain that is structurally conserved in several other proteins. This protein also contained a proline-rich region and a proline- and glutamine-rich region. The expression of Hrs mRNA was detected in all adult mouse tissues tested and also in embryos. To analyze the Hrs cDNA product, we prepared a polyclonal antibody against bacterially expressed Hrs. Using this antibody, we showed by subcellular fractionation that Hrs is localized to the cytoplasm; we also showed that that tyrosine phosphorylation of Hrs is induced in cells treated with epidermal growth factor or platelet-derived growth factor. These results suggest that Hrs plays a unique and important role in the signaling pathway of growth factors.     

Lymphocyte lineage-restricted tyrosine-phosphorylated proteins that bind PLC gamma 1 SH2 domains.

Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibroblasts induces tyrosine phosphorylation of PLC gamma 1 and stable association of PLC gamma 1 with the receptor protein tyrosine kinase. Similarly in lymphocytes, cross-linking of antigen receptors induces the formation of molecular complexes incorporating PLC gamma 1; however, associated kinase activity is thought to be mediated through cytoplasmic protein tyrosine kinase(s). In this report, we generated a fusion protein containing the SH2 domains of human PLC gamma 1 and human IgG1 heavy chain constant region to identify lymphocyte phosphoprotein-binding PLC gamma 1 SH2 domains following cellular activation. As in EGF- or platelet-derived growth factor-stimulated fibroblasts, PLC gamma 1 is coprecipitated in activated lymphocytes, complexed with associated tyrosine-phosphorylated proteins. One of these, a 35/36-kDa protein found prominently in T cells and at lower levels in B cells, bound to the fusion protein in immunoprecipitation experiments. The fusion protein showed lineage restricted association with a 74-kDa phosphoprotein in T cells and a 93-kDa phosphoprotein in B cells. It bound to activated EGF receptor in fibroblasts as expected, and protein tyrosine kinase activity was precipitated from EGF-stimulated cells. However, PLC gamma 1-associated protein tyrosine kinase activity was not detected in activated lymphocytes. These data suggest that lymphocyte PLC gamma 1 SH2-binding proteins are cell lineage specific and may be transiently associated with activated PLC gamma 1.     

Purification and characterization of phosphoinositide 3-kinase from rat liver

Phosphoinositide 3-kinase was purified 27,000-fold from rat liver. The enzyme was purified by acid precipitation of the cytosol followed by chromatography on DEAE-Sepharose, S-Sepharose, hydroxylapatite, Mono-Q, and Mono-S columns. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified phosphoinositide 3-kinase preparation contained an 85-kDa protein and a protein doublet of approximately 110 kDa. The 85- and 110-kDa proteins focus together on native isoelectric focusing gels and are cross-linked by dithiobis(succinylamide propionate), showing that the 110- and 85-kDa proteins are a complex. The apparent size of the native enzyme, as determined by gel filtration, is 190 kDa. The 85-kDa subunit is the same protein previously shown to associate with polyoma virus middle T antigen and the platelet-derived growth factor receptor (Kaplan, D. R., Whitman, M., Schaffhausen, B., Pallas, D. C., White, M., Cantley, L., and Roberts, T. M. (1987) Cell 50, 1021-1029). The two proteins co-migrate on two-dimensional gels; and, using a Western blotting procedure, 32P-labeled middle T antigen specifically blots the 85-kDa protein. The purified enzyme phosphorylates phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. The apparent Km values for ATP were found to be 60 microM with phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate as the substrate. The apparent Km for phosphatidyinositol is 60 microM, for phosphatidylinositol 4-phosphate is 9 microM, and for phosphatidylinositol 4,5-bisphosphate is 4 microM. The maximum specific activity using phosphatidylinositol as the substrate is 0.8 mumol/mg/min. The enzyme requires Mg2+ with an optimum of 5 mM. Substitution of Mn2+ for Mg2+ results in only approximately 10% of the Mg2(+)-dependent activity. Physiological calcium concentrations have no effect on the enzyme activity. Phosphoinositide 3-kinase has a broad pH optimum around 7.     

A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase.

Autophosphorylated growth factor receptors provide binding sites for the src homology 2 domains of intracellular signaling molecules. In response to epidermal growth factor (EGF), the activated EGF receptor binds to a complex containing the signaling protein GRB2 and the Ras guanine nucleotide-releasing factor Sos, leading to activation of the Ras signaling pathway. We have investigated whether the platelet-derived growth factor (PDGF) receptor binds GRB2-Sos. In contrast with the EGF receptor, the GRB2 does not bind to the PDGF receptor directly. Instead, PDGF stimulation induces the formation of a complex containing GRB2; 70-, 80-, and 110-kDa tyrosine-phosphorylated proteins; and the PDGF receptor. Moreover, GRB2 binds directly to the 70-kDa protein but not to the PDGF receptor. Using a panel of PDGF beta-receptor mutants with altered tyrosine phosphorylation sites, we identified Tyr-1009 in the PDGF receptor as required for GRB2 binding. Binding is inhibited by a phosphopeptide containing a YXNX motif. The protein tyrosine phosphatase Syp/PTP1D/SHPTP2/PTP2C is approximately 70 kDa, binds to the PDGF receptor via Tyr-1009, and contains several YXNX sequences. We found that the 70-kDa protein that binds to the PDGF receptor and to GRB2 comigrates with Syp and is recognized by anti-Syp antibodies. Furthermore, both GRB2 and Sos coimmunoprecipitate with Syp from lysates of PDGF-stimulated cells, and GRB2 binds directly to tyrosine-phosphorylated Syp in vitro. These results indicate that GRB2 interacts with different growth factor receptors by different mechanisms and the cytoplasmic phosphotyrosine phosphatase Syp acts as an adapter between the PDGF receptor and the GRB2-Sos complex.     

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.     

Regulation of the interleukin 2 receptor complex tyrosine kinase activity in vitro.

Interleukin 2 (IL-2) has been shown to stimulate tyrosine phosphorylation of a number of proteins requiring only the p75 beta chain of the IL-2 receptor. Unlike the receptors for epidermal growth factor, insulin, and other growth factors, the p55-alpha and p75-beta chains of the IL-2 receptor have no tyrosine protein kinase domain suggesting that the IL-2 receptor complex activates protein kinases by a unique mechanism. The activation of tyrosine kinases by IL-2 in situ was studied and using a novel methodology has shown tyrosine kinase activity associated with the purified IL-2R complex in vitro. IL-2 stimulated the in situ tyrosine phosphorylation of 97 kDa and 58 kDa proteins which bound to poly(Glu,Tyr)4:1, a substrate for tyrosine protein kinases, suggesting these proteins had characteristics found in almost all tyrosine kinases. IL-2 was found to stimulate tyrosine protein kinase activity in receptor extracts partially purified from human T lymphocytes and the YT cell line. Biotinylated IL-2 was used to precipitate the high-affinity-receptor complex and phosphoproteins associated with it. The data indicated that the 97-kDa and 58-kDa phosphotyrosyl proteins were tightly associated with the IL-2 receptor complex. These proteins were phosphorylated on tyrosine residues by IL-2 stimulation of intact cells and ligand treatment of in vitro receptor extracts. Furthermore, the 97-kDa and 58-kDa proteins were found in streptavidin-agarose/biotinylated IL-2 purified receptor preparations and showed high affinity for tyrosine kinase substrate support matrixes. The experiments suggest that these two proteins are potential candidates for tyrosine kinases involved in the IL-2R complex signal transduction process.     

Synthesis and assembly of a functionally active recombinant platelet-derived growth factor AB heterodimer

A Chinese hamster ovary cell line that stably expresses transfected human platelet-derived growth factor (PDGF) A and B chain precursors was established. All three dimeric combinations of PDGF chains were produced by this cell line; their biosynthesis, assembly, and processing were followed by pulse-chase analyses. PDGF-AA, PDGF-AB, and PDGF-BB were processed to Mr values of about 30,000 and were accumulated in these forms in the medium. In addition, PDGF-BB was further processed to a 24-kDa component, which remained cell-associated. The major secreted component was PDGF-AB, which was purified and shown to have structural and functional characteristics indistinguishable from PDGF-AB purified from human platelets.