A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains.

Endothelial cell surfaces play key roles in several important physiological and pathological processes such as blood clotting, angiogenic responses, and inflammation. Here we describe the cloning and characterization of tie, a novel type of human endothelial cell surface receptor tyrosine kinase. The extracellular domain of the predicted tie protein product has an exceptional multidomain structure consisting of a cluster of three epidermal growth factor homology motifs embedded between two immunoglobulinlike loops, which are followed by three fibronectin type III repeats next to the transmembrane region. Additionally, a cDNA form lacking the first of the three epidermal growth factor homology domains was isolated, suggesting that alternative splicing creates different tie-type receptors. Cells transfected with tie cDNA expression vector produce glycosylated polypeptides of 117 kDa which are reactive to antisera raised against the tie carboxy terminus. The tie gene was located in chromosomal region 1p33 to 1p34. Expression of the tie gene appeared to be restricted in some cell lines; large amounts of tie mRNA were detected in endothelial cell lines and in some myeloid leukemia cell lines with erythroid and megakaryoblastoid characteristics. In addition, mRNA in situ studies further indicated the endothelial expression of the tie gene. The tie receptor tyrosine kinase may have evolved for multiple protein-protein interactions, possibly including cell adhesion to the vascular endothelium.     

Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains

Epidermal growth factor (EGF) regulates cell proliferation and differentiation by binding to the EGF receptor (EGFR) extracellular region, comprising domains I-IV, with the resultant dimerization of the receptor tyrosine kinase. In this study, the crystal structure of a 2:2 complex of human EGF and the EGFR extracellular region has been determined at 3.3 A resolution. EGFR domains I-III are arranged in a C shape, and EGF is docked between domains I and III. The 1:1 EGF*EGFR complex dimerizes through a direct receptor*receptor interaction, in which a protruding beta-hairpin arm of each domain II holds the body of the other. The unique "receptor-mediated dimerization" was verified by EGFR mutagenesis.     

N-acetylcysteine inhibits angiotensin ii-mediated activation of extracellular signal-regulated kinase and epidermal growth factor receptor

Angiotensin II (Ang II) is known to stimulate reactive oxygen species (ROS) generation and epidermal growth factor (EGF) receptor transactivation to mediate growth-promoting signals such as extracellular signal-regulated kinase (ERK) in vascular smooth muscle cells (VSMCs). However, how ROS and EGF receptor interact to orchestrate these signals in VSMCs remains unclear. Here we found that an antioxidant, N-acetylcysteine, inhibited ERK activation and EGF receptor tyrosine phosphorylation induced by Ang II. Moreover, H(2)O(2) stimulates EGF receptor tyrosine phosphorylation and EGF receptor inhibitors attenuated H(2)O(2)-induced ERK activation. These data indicate that ROS mediate Ang II-induced EGF receptor transactivation, a critical mechanism for ERK-dependent growth in VSMCs.     

Bipartite inhibition of Drosophila epidermal growth factor receptor by the extracellular and transmembrane domains of Kekkon1

In Drosophila, signaling by the epidermal growth factor receptor (EGFR) is required for a diverse array of developmental decisions. Essential to these decisions is the precise regulation of the receptor's activity by both stimulatory and inhibitory molecules. To better understand the regulation of EGFR activity we investigated inhibition of EGFR by the transmembrane protein Kekkon1 (Kek1). Kek1 encodes a molecule containing leucine-rich repeats (LRR) and an immunoglobulin (Ig) domain and is the founding member of the Drosophila Kekkon family. Here we demonstrate with a series of Kek1-Kek2 chimeras that while the LRRs suffice for EGFR binding, inhibition in vivo requires the Kek1 juxta/transmembrane region. We demonstrate directly, and using a series of Kek1-EGFR chimeras, that Kek1 is not a phosphorylation substrate for the receptor in vivo. In addition, we show that EGFR inhibition is unique to Kek1 among Kek family members and that this function is not ligand or tissue specific. Finally, we have identified a unique class of EGFR alleles that specifically disrupt Kek1 binding and inhibition, but preserve receptor activation. Interestingly, these alleles map to domain V of the Drosophila EGFR, a region absent from the vertebrate receptors. Together, our results support a model in which the LRRs of Kek1 in conjunction with its juxta/transmembrane region direct association and inhibition of the Drosophila EGFR through interactions with receptor domain V.     

Elastase-released epidermal growth factor recruits epidermal growth factor receptor and extracellular signal-regulated kinases to down-regulate tropoelastin mRNA in lung fibroblasts

Elastase/anti-elastase imbalance is a hallmark of emphysema, a chronic obstructive pulmonary disease associated with the rupture and inefficient repair of interstitial elastin. We report that neutrophil elastase (NE) at low physiologic concentrations, ranging from 35 nm to 1 microm, invokes transient, peaking at 15 min, activation of extracellular signal-regulated kinases 1 and 2 (ERK) in elastogenic lung fibroblasts. ERK activation is preceded by the release of soluble 25-26-kDa forms of epidermal growth factor (EGF) and transactivation of EGF receptor (EGFR) in NE-exposed cells. The stimulatory effect of NE on ERK is abrogated in the presence of anti-EGF-neutralizing antibodies, EGFR tyrosine kinase inhibitor (AG1478), and ERK kinase inhibitor (PD98059), as well as abolished in both EGFR-desensitized and endocytosis-arrested fibroblasts. Nuclear accumulation of activated ERK is associated with transient, peaking at 30 min, induction of c-Fos and sustained, observed at 24-48 h, decrease of tropoelastin mRNA levels in NE-challenged cells. Pretreatment of fibroblasts with AG1478 or PD98059 abrogates the NE-initiated tropoelastin mRNA suppression. We conclude that proteolytically released EGF signals directly via EGFR and ERK to down-regulate tropoelastin mRNA in NE-challenged lung fibroblasts.     

Molecular imaging of epidermal growth factor receptor kinase activity

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is commonly altered in different tumor types, leading to abnormally regulated kinase activity and excessive activation of downstream signaling cascades, including cell proliferation, differentiation, and migration. To investigate the EGFR signaling events in real time and in living cells and animals, here we describe a multidomain chimeric reporter whose bioluminescence can be used as a surrogate for EGFR kinase activity. This luciferase-based reporter was developed in squamous cell carcinoma cells (UMSCC1) to generate a cancer therapy model for imaging EGFR. The reporter is designed to act as a phosphorylated substrate of EGFR and reconstitutes luciferase activity when it is not phosphorylated, thereby providing a robust indication of EGFR inhibition. We validated the reporter in vitro and demonstrated that its activity could be differentially modulated by EGFR tyrosine kinase inhibition with erlotonib or receptor activation with epidermal growth factor. Further experiments in vivo demonstrated quantitative and dynamic monitoring of EGFR tyrosine kinase activity in xenograft. Results obtained from these studies provide unique insight into pharmacokinetics and pharmacodynamics of agents that modulate EGFR activity, revealing the usefulness of this reporter in evaluating drug availability and cell targeting in both living cells and mouse models.     

Suppression of protein tyrosine kinase activity of the epidermal growth factor receptor by epidermal growth factor

Epidermal growth factor (EGF) receptor protein kinase activity, estimated by the use of peptide substrates, was reduced by as much as 70% after the treatment of intact A431 human carcinoma cells with EGF. The apparent decrease in protein kinase activity was observed after immunoprecipitation of the receptor or after purification of the receptor by lectin chromatography. By the use of [35S]methionine, it was determined that the total amount of receptor obtained was the same whether or not cells were treated with EGF. EGF stimulated the purified receptor protein kinase activity in vitro; however, the EGF-stimulated activity of receptor from EGF-treated cells continued to be reduced by as much at 70% compared to the EGF-stimulated activity from untreated cells. The reduction in receptor protein kinase activity induced by EGF may represent a feedback mechanism by which responsiveness to the growth factor is regulated.     

Crystal structure of a truncated epidermal growth factor receptor extracellular domain bound to transforming growth factor alpha

We report the crystal structure, at 2.5 A resolution, of a truncated human EGFR ectodomain bound to TGFalpha. TGFalpha interacts with both L1 and L2 domains of EGFR, making many main chain contacts with L1 and interacting with L2 via key conserved residues. The results indicate how EGFR family members can bind a family of highly variable ligands. In the 2:2 TGFalpha:sEGFR501 complex, each ligand interacts with only one receptor molecule. There are two types of dimers in the asymmetric unit: a head-to-head dimer involving contacts between the L1 and L2 domains and a back-to-back dimer dominated by interactions between the CR1 domains of each receptor. Based on sequence conservation, buried surface area, and mutagenesis experiments, the back-to-back dimer is favored to be biologically relevant.     

Proteolytic domains of the epidermal growth factor receptor of human placenta

Microsomal membranes from human placenta, which bind 5–20 pmol of ¹²⁵I-epidermal growth factor (EGF) per mg protein, have been affinity-labeled with ¹²⁵I-EGF either spontaneously or with dimethylsuberimidate. Coomassie blue staining patterns on SDS polyacrylamide gels are minimally altered, and the EGF-receptor complex appears as a specifically labeled band of 180,000 daltons which is not removed by urea, neutral buffers, or chaotropic salts but is partially extracted by mild detergents. Limited proteolysis by alpha chymotrypsin and several other serine proteases yields labeled fragments of 170,000, 130,000, 85,000, and 48,000 daltons. More facile cleavage by papain or bromelain rapidly degrades the hormone-receptor complex to smaller labeled fragments of about 35,000 and 25,000 daltons. These fragments retain the binding site for EGF, are capable of binding EGF, and remain associated with the membrane. Alpha chymotryptic digestion of receptor solubilized by detergents yields the same fragments obtained with intact vesicles, suggesting that the fragments may represent intrinsic proteolytic domains of the receptor.     

Site-specific antibodies to the erbB oncogene product immunoprecipitate epidermal growth factor receptor

Antiserum to a defined region (residues 373-383) of the erbB oncogene product immunoprecipitated a 170,000 dalton protein that was phosphorylated in an EGF-sensitive fashion as well as the 125I-EGF-receptor complex from A431 human epidermoid carcinoma cells. Preincubation of the antiserum with an excess of the synthetic peptide corresponding to the defined region blocked the immunoprecipitation of this protein. A partial proteolytic peptide map of this immunoprecipitated 170,000 dalton protein was identical to that of the authentic EGF receptor. These results suggest immunological similarity between the erbB gene product and the EGF receptor.     

In-depth biophysical analysis of interactions between therapeutic antibodies and the extracellular domain of the epidermal growth factor receptor

Targeting of the epidermal growth factor receptor (EGFR) with monoclonal antibodies has become an established antitumor strategy in clinical use or in late stages of drug development. The mAbs effector mechanisms have been widely analyzed based on in vivo or cell studies. Hereby we intend to complement these functional studies by investigating the mAb-EGFR interactions on a molecular level. Surface plasmon resonance, isothermal titration calorimetry, and static light scattering were employed to characterize the interactions of matuzumab, cetuximab, and panitumumab with the extracellular soluble form ecEGFR. The kinetic and thermodynamic determinants dissected the differences in mAbs binding mechanism toward ecEGFR. The quantitative stoichiometric data clearly demonstrated the bivalent binding of the mAbs to two ecEGFR molecules. Our results complement earlier studies on simultaneous binding of cetuximab and matuzumab. The antibodies retain their bivalent binding mode achieving a 1:2:1 complex formation. Interestingly the binding parameters remain nearly constant for the individual antibodies in this ternary assembly. In contrast the binding of panitumumab is almost exclusive either by directly blocking the accessibility for the second antibody or by negative allosteric modulation. Overall we provide a comprehensive biophysical dataset on binding parameters, the complex assembly, and relative epitope accessibility for therapeutic anti-EGFR antibodies.     

Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation

The possible role of epidermal growth factor (EGF) receptor phosphorylation at threonine 654 in modulating the protein-tyrosine kinase activity of EGF-treated A431 cells has been studied. It has been suggested that EGF could indirectly activate a protein-serine/threonine kinase, protein kinase C, that can phosphorylate the EGF receptor at threonine 654. Protein kinase C is known to be activated, and threonine 654 is phosphorylated, when A431 cells are exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The protein-tyrosine kinase activity of EGF receptors is normally evidenced in EGF-treated cells by phosphorylation of the receptor at tyrosine. This is inhibited when TPA-treated cells are exposed to EGF. We now show that receptor phosphorylation at threonine 654 can also be detected in EGF-treated A431 cells, presumably due to indirect stimulation of protein kinase C or a similar kinase. Some receptor molecules are phosphorylated both at threonine 654 and at tyrosine. Since prior phosphorylation at threonine 654 inhibits autophosphorylation, we propose that protein kinase C can phosphorylate the threonine 654 of autophosphorylated receptors. This provides evidence for models in which protein kinase C activation, consequent upon EGF binding, could reduce the protein-tyrosine kinase activity of the EGF receptor. Indeed, we find that 12-O-tetradecanoylphorbol-13-acetate, added 10 min after EGF, further increases threonine 654 phosphorylation and induces the loss of tyrosine phosphate from A431 cell EGF receptors.     

Ratiometric assay of epidermal growth factor receptor tyrosine kinase activation

Activation of cells is frequently followed by tyrosine phosphorylation of proteins. To quantify this process, we developed a ratiometric enzyme-linked immunosorbent assay (ELISA) using epidermal growth factor receptors (EGFR) as a model. Microtiter dishes were coated with anti-EGFR monoclonal antibodies to capture the receptor followed by parallel detection of receptor and phosphotyrosine content with secondary antibodies. The ratio of these two parameters was found to directly reflect EGFR activation and was insensitive to the effect of receptor downregulation. Our assay could resolve differences in EGFR activation due to small changes (less than 1 ng/ml) in ligand. We found that phosphotyrosine detection by ELISA was 8- to 32-fold more sensitive than Western blot detection and could be reliably detected using as little as 4 ng of cellular lysate. Detection of EGFR levels by ELISA was 30 times more sensitive than Western blot analysis and was reliable for as low as 8 ng of cellular lysate per well. Because of the wide linear range of the ELISA, we could directly compare receptor activation in cell types with different EGFR expression levels. Our assay provides a rapid and sensitive method of determining EGFR activation status and could be easily modified to evaluate any tyrosine-phosphorylated protein.     

Platelet-derived growth factor requires epidermal growth factor receptor to activate p21-activated kinase family kinases

The platelet-derived growth factor (PDGF) receptor (PDGFR) transactivates the epidermal growth factor (EGF) receptor (ErbB1) to stimulate the cell migration of fibroblasts through an unknown mechanism (Li, J., Kim, Y. N. & Bertics, P. (2000) J. Biol. Chem. 275, 2951-2958). In this paper we provide evidence that the transactivation of the EGF receptor (EGFR) by PDGFR is essential for PDGF to activate p21-activated kinase (PAK) family kinases. Fetal calf serum (10%) transiently stimulates the PAK activity in NIH 3T3 fibroblasts. The activation of PAK was completely inhibited by either PDGFR-specific inhibitor (AG1295) or EGFR-specific inhibitor (AG1478), suggesting that serum requires either the PDGF- or EGF-dependent pathway or the combination of both to activate PAK. PDGF-induced activation of PAK is completely inhibited by either AG1295 or AG1478, indicating that PDGF requires both PDGFR and EGFR for PAK activation. In support of this notion, a mouse embryo fibroblast cell line derived from the EGFR -/- mouse (from Dr. Erwin Wagner) doesn't activate PAK in response to PDGF. Expression of human EGFR in this cell line restores the ability of the PDGF to induce PAK activation. Our results indicate that PDGF activates PAK through transactivation of ErbB1.     

Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase

The relationship between epidermal growth factor receptor (EGF-R) protein tyrosine kinase activation and ligand-induced receptor dimerization was investigated using several bivalent anti-EGF-R antibodies directed against various receptor epitopes. In A431 membrane preparations and permeabilized cells, all antibodies were able to activate the EGF-R tyrosine kinase, as measured by EGF-R autophosphorylation and phosphorylation of other substrates on tyrosine residues. EGF-R tyrosine kinase activation correlated strongly with the induction of EGF-R dimerization. (i) Both processes specifically occurred in a narrow antibody concentration range; (ii) both processes required the presence of detergent; and (iii) both processes depended on antibody bivalence since monovalent Fab fragments were inactive yet regained full activity after cross-linking by a second bivalent antibody. These data demonstrate that antibody bivalence is essential and sufficient for EGF-R activation and that activation occurs regardless of the EGF-R epitope recognized. Finally, EGF-R dimerization was shown not to depend on receptor autophosphorylation since it still occurred in the absence of ATP. Also, partial inhibition of the tyrosine kinase activity by the specific EGF-R tyrosine kinase inhibitor tyrphostin AG 213 did not affect formation of EGF-R dimers. Taken together these results demonstrate that induction of EGF-R dimerization is sufficient and in case of antibody action, essential, for activation of the EGF-R tyrosine kinase and thus provide strong support for an intermolecular mechanism of EGF-R tyrosine kinase activation.     

The epidermal growth factor receptor tyrosine kinase phosphorylates connexin32

Oncoprotein 18 or stathmin was isolated from bovine brain, characterized and novel features of its function as a microtubule depolymerizing factor were tested.The effect of phosphorylation of stathmin on its function as a microtubule depolymerizing factor has been tested in vitro. Five different protein kinases, protein kinase A, MAP kinase, cdc2 kinase, glycogen synthase kinase 3 and casein kinase 2, were used to modify stathmin, since it is known that these kinases could phosphorylate several residues that are modified in vivo and could have important roles in stathmin function. The residues phosphorylated in vitro by the different protein kinases were identified and in some cases they correspond to those modified in vivo.Recombinant unphosphorylated stathmin and native stathmin, which was previously dephosphorylated with alkaline phosphatase, showed similar microtubule depolymerizing activity. This activity is higher than that of stathmin phosphorylated by protein kinase A, MAP kinase or cdc 2 kinase, whereas phosphorylation of the protein with casein kinase 2 or glycogen synthase kinase 3 resulted in a slight increase of the depolymerizing activity.     

Calmodulin inhibits the epidermal growth factor receptor tyrosine kinase.

We demonstrate in this report that the epidermal growth factor (EGF) receptor from rat liver can be isolated by calmodulin affinity chromatography by binding in the presence of Ca2+ and elution with a Ca(2+)-chelating agent. The bulk of the EGF receptor is not eluted by a NaCl gradient in the presence of Ca2+. We ascertained the identity of the isolated receptor by immunoblot and immunoprecipitation using a polyclonal antibody against an EGF receptor from human origin. The purified receptor is autophosphorylated in tyrosine residues in an EGF-stimulated manner, and EGF-dependent phosphorylation of serine residues was also detected. Both the EGF and the transforming growth factor-alpha stimulate the tyrosine-directed protein kinase activity of the isolated receptor with similar affinities. Furthermore, we demonstrate that calmodulin inhibits the EGF-dependent tyrosine-directed protein kinase activity associated to the receptor in a concentration-dependent manner. This inhibition is partially Ca2+ dependent and is not displaced by increasing the concentration of EGF up to an EGF/calmodulin ratio of 10 (mol/mol). In addition, calmodulin was phosphorylated in an EGF-stimulated manner in the presence of a basic protein (histone) as cofactor and in the absence, but not in the presence, of Ca2+.