Signal transduction by vascular endothelial growth factor receptors

VEGFs (vascular endothelial growth factors) control vascular development during embryogenesis and the function of blood vessels and lymphatic vessels in the adult. There are five related mammalian ligands, which act through three receptor tyrosine kinases. Signalling is modulated through neuropilins, which act as VEGF co-receptors. Heparan sulfate and integrins are also important modulators of VEGF signalling. Therapeutic agents that interfere with VEGF signalling have been developed with the aim of decreasing angiogenesis in diseases that involve tissue growth and inflammation, such as cancer. The present review will outline the current understanding and consequent biology of VEGF receptor signalling.     

Signal transduction by tumor necrosis factor receptors

Tumor necrosis factor (TNF) is a key mediator in the inflammatory response which is implicated in the onset of a number of diseases. Research on TNF led to the characterization of the largest family of cytokines known until now, the TNF superfamily, which exert their biological effects through the interaction with transmembrane receptors of the TNFR superfamily. TNF itself exerts its biological effects interacting with two different receptors: TNFR1 and TNFR2. TNFR1 presents a death domain on its intracellular region. In contrast to TNFR1, TNFR2 does not have a death domain. Activation of TNFR1 implies the consecutive formation of two different TNF receptor signalling complexes. Complex I controls the expression of antiapoptotic proteins that prevent the triggering of cell death processes, whereas Complex II triggers cell death processes. TNFR2 only signals for antiapoptotic reactions. However, recent evidence indicates that TNFR2 also signals to induce TRAF2 degradation. TRAF2 is a key mediator in signal transduction of both TNFR1 and TNFR2. Thus, this novel signalling pathway has two important implications: on one hand, it represents an auto regulatory loop for TNFR2; on the other hand, when this signal is triggered TNFR1 activity is modified so that antiapoptotic pathways are inhibited and apoptotic reactions are enhanced.     

Reconstitution of the high affinity epidermal growth factor receptor on cell-free membranes after transmodulation by platelet-derived growth factor

We have prepared plasma membranes from Balb/c 3T3 fibroblasts to study the transmodulation of the high affinity epidermal growth factor (EGF) receptor. Although phorbol esters do not transmodulate the high affinity EGF receptors on these membranes, the addition of platelet-derived growth factor (PDGF) or EGF to the membranes leads to the loss of high affinity EGF binding and to the phosphorylation of several membrane proteins, including the EGF receptor. The EGF receptor is phosphorylated at tyrosine residues although we have not yet established if this represents direct phosphorylation by the PDGF receptor kinase or is mediated by activation of other cell membrane-associated tyrosine kinases. Upon treatment of the membranes with PDGF, four major phosphoproteins (of apparent molecular masses of 69, 56, 38, and 28 kDa) are released from the membrane and can be retrieved from the supernatant fluid using a reversed-phase cartridge. As assessed by immunoprecipitation with an anti-phosphotyrosine antibody, all four proteins appear to be phosphorylated on tyrosine. The time course of dissociation of these proteins from the membranes closely parallels the loss of high affinity EGF receptors. The high affinity EGF receptor can be reconstituted on PDGF-transmodulated membranes by treating the supernatant fluid with alkaline phosphatase and adding the mixture to the membranes. It appears that dephosphorylation of the released proteins is sufficient to allow reassociation with the membranes and formation of the high affinity EGF receptor complex.     

Signal transduction by the epidermal growth factor receptor is attenuated by a COOH-terminal domain serine phosphorylation site.

It has been proposed that the acute desensitization of epidermal growth factor receptor (EGF-R) function can be accounted for, in part, by the effect of EGF to increase phosphorylation of the receptor at Ser1046/7 (Countaway, J.L., Nairn, A.C., and Davis, R.J. (1992) J. Biol. Chem. 267, 1129-1140). Here, we show that the mutational removal of this phosphorylation site causes an activation of EGF-R function and a potentiation of signal transduction. The mechanism of potentiation results from 1) defective down-regulation of the EGF-R when cells are incubated with high concentrations of EGF; and 2) increased EGF-stimulated tyrosine phosphorylation. The increased EGF-stimulated phosphorylation is associated with an alteration of the apparent specificity of tyrosine phosphorylation and is independent of the down-regulation defect. Together, these data strongly support the hypothesis that Ser1046/7 is a biologically significant site of regulatory phosphorylation of the EGF-R.     

Nerve growth factor mediates signal transduction through trk homodimer receptors.

We have investigated the molecular nature of the high affinity nerve growth factor (NGF) receptors by using cell lines expressing gp75LNGFR and gp140trk. Our results suggest that gp75LNGFR and gp140trk interact with NGF independently and that only gp140trk mediates NGF signaling. NGF binds to gp140trk with picomolar affinity and induces its phosphorylation on tyrosine residues regardless of the presence of gp75LNGFR. NGF-gp140trk complexes display the slow dissociation rate and rapid internalization characteristics of high affinity NGF receptors. Cross-linking studies reveal the existence of gp75LNGFR and gp140trk homodimers. However, we were unable to detect gp75LNGFR-gp140trk heterodimers. Coexpression in COS cells of wild-type and kinase deficient mutants reveals that gp140trk receptors can undergo intermolecular phosphorylation, indicating the formation of functional homodimers. Moreover, these kinase deficient mutants inhibit NGF-induced signaling through wild-type gp140trk receptors. These results indicate that the functional high affinity NGF receptors consist of gp140trk homodimeric (or oligomeric) complexes.     

Evidence that biological activity of NGF is mediated through a novel subclass of high affinity receptors.

Trophic factors, such as NGF, regulate survival and differentiation of many classes of neurons by binding specific receptors. Two types of NGF receptors have been identified, which bind NGF with low and high affinity. The latter mediates the major biological actions of NGF. To determine the relationship between these two receptor types, polyclonal antibodies to the low affinity receptor have been prepared and used in ligand-binding, ligand-cross-linking, and biological assays. These antibodies eliminated binding of NGF to low affinity receptors and to one class of high affinity receptors, but did not prevent binding to a second class of high affinity receptors. The same antibodies did not inhibit NGF-stimulated neuronal survival or neurite outgrowth. Thus, a biologically important class of high affinity NGF receptors is antigenically distinct from the low affinity receptor and may be encoded by a novel gene.     

Human esophageal carcinoma cells have fewer, but higher affinity epidermal growth factor receptors

Squamous cell carcinomas have recently been shown to contain increased numbers of epidermal growth factor (EGF) receptors. Since EGF has an important role in epithelial growth and differentiation, it is possible that modulation of its receptor may have an important role in neoplasia. In an attempt to further explore the relationship of EGF receptor expression to malignant transformation, we examined 14 squamous cell carcinoma cell lines of the esophagus for the number and affinity of EGF receptors. Seven cell lines were newly isolated by this laboratory and recently characterized. The seven additional cell lines were obtained from Japan (4 cell lines) and South Africa (3 cell lines). Surprisingly, we found that esophageal carcinomas contained lowered quantities of surface EGF receptors (2- to 100-fold) and that the affinity of the EGF receptor was increased (6- to 100-fold) when compared to normal esophageal epithelial cells. Moreover, the biologic response of esophageal carcinoma cells to EGF differed markedly from that of other squamous cell tumor cells exhibiting elevated numbers of receptors, such as A431 and SCC-15. Human esophageal carcinoma cells were maximally stimulated by the addition of 5 ng/ml of EGF, similar to normal esophageal keratinocytes, but in contrast to normal cells were not inhibited by the higher concentrations tested (up to 40 ng/ml). On the other hand, addition of any EGF to the medium (beyond that normally present in serum) was found to dramatically inhibit the growth of A431 and SCC-15 cells. Our findings indicate that squamous cell neoplasia is not dependent upon increased numbers of cell surface EGF receptors, that EGF receptor number may have a determinant role in EGF cell toxicity, and that the stimulatory response of cells to EGF may reflect a complex function of EGF receptor number, affinity, and occupancy.     

Cyclic AMP potentiates down regulation of epidermal growth factor receptors by platelet-derived growth factor

Pretreatment of $\text{Balb}\text{c}\text{-3T3}$ cells with platelet-derived growth factor (PDGF) has been shown to decrease binding sites for ¹²⁵I-labelled epidermal growth factor (EGF) (1,2,3). Agents which elevate cellular cyclic AMP concentrations enhance this ability, and the change in EGF binding is inversely proportional to the elevation of cyclic AMP. In quiescent density arrested cells, the sensitivity of cells to down regulation of EGF receptors by PDGF is proportional to the cyclic AMP content of the cultures in three different cell lines. Agents which elevate cyclic AMP and which potentiate PDGF mediated heterologous down regulation of EGF receptors are able, like cholera toxin (3), to stimulate cells to synthesize DNA in defined medium in the absence of EGF. Down regulation of EGF receptors by PDGF in combination with agents elevating cyclic AMP effectively mimics the action of EGF.     

Production of a biologically active epidermal growth factor fusion protein with high collagen affinity

Collagen is generally incapable of capturing polypeptides such as growth factors in a specific manner. In this study, we established a collagen-binding growth factor (FNCBD-EGF) consisting of epidermal growth factor (EGF) and the fibronectin collagen-binding domain. A typical yield of FNCBD-EGF was approximately 200 microg/ml culture in an Escherichia coli expression system. This fusion protein bound to gelatin and fibrillar collagen sponges, and the bound protein was not effectively eluted even with 2 M NaCl. In addition, FNCBD-EGF bound to type I, II, III, or IV collagen-coated plates, and the specificity of binding was confirmed by competitive inhibition using fibronectin. FNCBD-EGF substantially stimulated cell growth after binding to collagen-coated culture plates, whereas EGF had no effect, indicating that this fusion protein acted as a collagen-associated growth factor. In an animal model of impaired wound healing, FNCBD-EGF, but not EGF, was retained with collagen sponges at wound sites 4 d after implantation, and repair of epidermis was observed underneath the sponges. These results suggested that our fusion protein with high collagen affinity would be useful for wound healing.     

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K_d 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.     

High affinity epidermal growth factor receptors on the surface of A431 cells have restricted lateral diffusion

Rhodamine-labelled epidermal growth factor (Rh-EGF) was shown to bind to A431 cells grown at low density both to a small number of high affinity receptors (KD = 2.8 X 10(-10) M; fraction of total binding sites approximately 0.12) and also to a large number of low affinity receptors (KD = 4 X 10(-9) M; fraction of total binding sites approximately 0.88). Measurements of the lateral diffusion of EGF receptors on the cell surface were made using Rh-EGF and the technique of fluorescence photobleaching recovery. The high affinity receptors (labelled with 1.6 X 10(-10) M Rh-EGF, 5% of EGF binding sites occupied) did not show lateral mobility over the temperature range 3 degrees-37 degrees C. The low affinity receptors (labelled with 2.4 X 10(-7) M Rh-EGF, 90% of EGF sites occupied) showed at least 75% fluorescence recovery after photobleaching, and lateral diffusion coefficients of approximately 2 X 10(-10) cm2/s. These results show that the two populations of EGF receptors defined by binding studies differ in their freedom to diffuse laterally. The observation that the high affinity receptors are immobile indicates that lateral diffusion of receptors, at least over a distance of a few hundred nanometres or more, may not be required for the action of low concentrations of EGF.     

Density-induced down regulation of epidermal growth factor receptors

Summary Previous studies have shown that cell density can regulate the binding of several growth factors. To determine whether cell density exerts a uniform effect on the expression of epidermal growth factor (EGF) receptors, seven cell lines were examined in detail. For each cell line, EGF binding was found to decrease as cell density increases. Scatchard analysis of the binding data reveals that decreases in EGF binding are due to reductions in the number of cell surface EGF receptors. The only apparent exception is the effect of cell density on the binding of EGF to A-431 cells. For these cells, increases in cell density lead to two effects: decreases in the number of high affinity EGF receptors and increases in the total number of EGF receptors. In addition to the effects of cell density on EGF receptors, it was determined that increases in cell density can coordinately down-regulate receptors for as many as four different growth factors. Overall, the findings described in this report for EGF and those previously described for transforming growth factor type-β (TGF-β) and fibroblast growth factor (FGF) demonstrate the existence of a common mechanism for down-regulating growth factor receptors. This work was supported by grants from the Nebraska Department of Health (89-51), the National Cancer Institute (Laboratory Research Center Support Grant, CA36727), and the American Cancer Society (Core Grant ACS SIG-16). EDITOR'S STATEMENT The paper by Rizzino et al. demonstrates that receptor number decreases as a function of cell density. This may represent a mechanism by which cell proliferation is reduced as cell density increases.     

Diacylglycerol treatment rapidly decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells

The synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol (OAG) and phorbol esters activate protein kinase C in intact cells. We report here that OAG inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The inhibition was detected as early as 1 min after treatment at 37 degrees C and persisted for at least 120 min. The effect of OAG was reversed upon removal of this diacylglycerol. Detailed Scatchard analysis of 125I-EGF binding to Swiss 3T3 cells at 4 degrees C after a 1 h incubation with a saturating dose of OAG at 37 degrees C, demonstrates that this OAG pretreatment does not change the apparent number of EGF receptors but causes a marked decrease in their apparent affinity for the ligand. Prolonged treatment (40 h) of the cells with phorbol dibutyrate (PBt2) which causes a marked decrease in the number of phorbol ester binding sites and in the activity of protein kinase C, prevented the inhibition of 125I-EGF binding by both PBt2 and OAG. The results support the possibility that protein kinase C plays a role in the transmodulation of the EGF receptor in intact cells.     

Dimerization of internalized epidermal growth factor receptors.

Binding of epidermal growth factor (EGF) to cell surface EGF receptors initiates the formation of the receptor homodimers that can be detected by covalent cross-linking in intact cells or in detergent-solubilized cell extracts. Low pH dissociation of EGF from surface receptors results in immediate monomerization of receptor dimers. Using chemical cross-linking during mild permeabilization or cell solubilization, we have detected dimers of internalized EGF receptors in human carcinoma A-431 cells and transfected NIH 3T3 cells that express human EGF receptors. The percentage of internalized cross-linked receptor dimers was similar to that observed for surface EGF receptors. Furthermore, at the time of maximal accumulation of EGF-receptor complexes within the endosomal compartment (10-15 min of incubation at 37 degrees C), both the dimeric and monomeric forms of the EGF receptor are tyrosine-phosphorylated to the same extent as surface dimer and monomer species. In transfected NIH 3T3 cells, the level of dimerized and internalized kinase-negative EGF receptors was not different from that observed for wild-type receptors. These data suggest that for some time after internalization EGF does not dissociate from its receptor and indicate that a receptor conformation is preserved intracellularly that allows maintenance of receptor-receptor interactions and tyrosine kinase activity.     

cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.     

Specific, high affinity relaxin-like factor receptors.

The relaxin-like factor (RLF) is a circulating hormone that binds to specific membrane-bound uterine receptors in the mouse. Mono-iodinated RLF tracers were produced and characterized specifically to study the properties of the RLF receptor. The tracers bound to the RLF receptor in uterine crude membrane preparations with high affinity (73 nM for (125)I-Tyr(A9) RLF and 90 nM for (125)I-Tyr(A26) RLF) as determined by Scatchard analysis. The specificity of binding was confirmed by chemical cross-linking experiments. Binding of (125)I-Tyr(A9) RLF to the putative receptor was inhibited in the presence of a 640-fold excess of unlabeled human RLF but not by the same excess of human relaxin. SDS-gel electrophoresis of the RLF-receptor complex revealed a molecular mass of >200 kDa, which remained unchanged upon reduction. The size and the lack of subunit structure of the receptor is similar to the features reported for the relaxin receptor. In this regard both, the RLF and the relaxin receptor are different from the insulin- and the insulin-like growth factor-type 1 receptors. This observation supports the relaxin-likeness of this new factor not only toward potential target tissues but also as regards receptor features.     

Regulation of epidermal growth factor receptor internalization by G protein-coupled receptors

The epidermal growth factor (EGF) receptor (EGFR) plays a central role in regulating cell proliferation, differentiation, and migration. Cellular responses to EGF are dependent upon the amount of EGFR present on the cell surface. Stimulation with EGF induces sequestration of the receptor from the plasma membrane and its subsequent downregulation. Recently, internalization of the EGFR was also shown to be required for mitogenic signaling via the activation of MAP kinases. Therefore, mechanisms regulating internalization of the EGFR represent an important facet for the control of cellular response. Here, we demonstrate that EGFR is removed from the cell surface not only following stimulation with EGF, but also in response to stimulation of G protein-coupled lysophosphatidic acid (LPA) and beta2 adrenergic (beta2AR) receptors. Using a FLAG epitope-tagged EGFR to quantitate receptor internalization, we show that incubation with EGF, LPA, or isoproterenol (ISO) causes the time-dependent loss of cell surface EGFR. Internalization of EGFR by these ligands involves the tyrosine kinase activity of the receptor itself and c-Src, as well as the GTPase activity of dynamin. Unexpectedly, we find that internalization of the EGFR by EGF is dependent upon Gbetagamma and beta-arrestin proteins; expression of minigenes encoding the carboxyl terminii of the G protein-coupled receptor kinase 2, or beta-arrestin1, attenuates LPA-, ISO-, and EGF-mediated internalization of EGFR. Thus, G protein-coupled receptors can control the function of the EGFR by regulating its endocytosis.     

Ligand-induced activation of A431 cell epidermal growth factor receptors occurs primarily by an autocrine pathway that acts upon receptors on the surface rather than intracellularly

A431 cells express high numbers of epidermal growth factor (EGF) receptors and produce a ligand for these receptors, transforming growth factor-alpha (TGF-alpha). We have obtained evidence that the EGF receptors on these cells may be activated through an "autocrine" pathway by ligand and have investigated whether activation of phosphorylation of the receptor by the endogenously produced TGF-alpha occurs intracellularly or at the cell surface. When A431 cells were cultured under serum-free conditions, in the absence of exogenous ligand, EGF receptors were found to have a basal level of phosphorylation. When cells were labeled by culturing with 32Pi in the continuous presence of monoclonal antibodies that block binding of TGF-alpha to the EGF receptor, phosphorylation decreased to 30 +/- 10% of the basal level. This reduction could not be accounted for by the decrease in receptor content attributable to down-regulation and catabolism of EGF receptors that resulted from the binding of anti-receptor monoclonal antibodies. The reduction in receptor phosphorylation mediated by antibody was accompanied by the accumulation of increased levels of secreted TGF-alpha species in the culture medium. We also pulse-labeled A431 cells for 15 min with [35S]cysteine and immunoprecipitated the cell lysate with anti-phosphotyrosine antibody after various chase periods. Tyrosine-phosphorylated EGF receptor became detectable after 40 min of chase and reached a maximum after 4-6 h; these times are in agreement with the intervals required for EGF receptors to reach the cell surface after synthesis and then to achieve maximal expression. In addition, only the 170-kDa, mature EGF receptor species, and not the 160-kDa intracellular precursor, was immunoprecipitated with the anti-phosphotyrosine antibody. The results of these pulse-chase experiments and the finding that anti-receptor monoclonal antibody can block receptor phosphorylation suggest that activation of EGF receptors can result from the binding of an endogenous ligand (presumably TGF-alpha), which occurs at the cell surface and not during receptor biosynthesis and intracellular processing.