Epidermal growth factor: the receptor and its function

Epidermal growth factor (EGF) is a small polypeptide hormone with mitogenic properties in vivo and in vitro. EGF elicits biologic responses by binding to a cell surface receptor which is a transmembrane glycoprotein containing a cytoplasmic protein tyrosine kinase. EGF responses are mediated by ligand binding and activation of this intrinsic protein kinase. The receptor can be phosphorylated by other protein kinases, and this may regulate receptor function. Stimulation of the receptor tyrosine kinase activity by ligand binding must regulate the activity of an as yet undefined molecule(s) responsible for transmitting a mitogenic signal to the nucleus.     

表皮生长因子受体及其变异性质

EGFR主要由三个结构区域组成,即EGF结合区域、跨膜区域和依赖EGF蛋白激酶区域。EGFR被激活后,不仅能使许多底物蛋白磷酸化,自身也发生磷酸化,这些磷酸化作用在信息传导中有重要作用。EGFR与EGF结合后,其复合体以胞饮方式内容形成受体小体,除部分被降解外,大部分在高尔基GERL区加工后到达细胞核调节基因表达。本文通过对受体激活模型和用体外遗传变异方法对变异EGFR的研究阐述了EGFR的结构与功能的关系。     

Epidermal growth factor stimulates tyrosine phosphorylation of human glucocorticoid receptor in cultured cells

Human breast epithelial HBL100 cells, which bind both epidermal growth factor (EGF) and glucocorticoids, were labelled to steady state specific activity with 32Pi and the glucocorticoid receptor was immunoprecipitated from cell lysates with polyclonal antiserum GR884. Immunoprecipitated receptor was resolved by NaDodSO4-polyacrylamide gel electrophoresis and identified by autoradiography. Immunoprecipitated receptor also was characterized by western blot analysis and affinity labelling with [3H]dexamethasone-21-mesylate. Phosphoamino acid analysis of 32P-glucocorticoid receptor revealed 89% phosphoserine and 11% phosphotyrosine. Treatment of steady state 32Pi-labelled cells with EGF stimulated total and alkali-stable phosphorylation in the 97 kDa receptor band by about 35%. Prior incubation with dexamethasone inhibited EGF stimulated, alkali-stable phosphorylation of the 97 kDa glucocorticoid receptor band.     

Epidermal growth factor induces tyrosine phosphorylation, membrane insertion, and activation of transient receptor potential channel 4

Various members of the canonical family of transient receptor potential channels (TRPCs) exhibit increased cation influx following receptor stimulation or Ca(2+) store depletion. Tyrosine phosphorylation of TRP family members also results in increased channel activity; however, the link between the two events is unclear. We report that two tyrosine residues in the C terminus of human TRPC4 (hTRPC4), Tyr-959 and Tyr-972, are phosphorylated following epidermal growth factor (EGF) receptor stimulation of COS-7 cells. This phosphorylation was mediated by Src family tyrosine kinases (STKs), with Fyn appearing to be the dominant kinase. In addition, EGF receptor stimulation induced the exocytotic insertion of hTRPC4 into the plasma membrane dependent on the activity of STKs and was accompanied by a phosphorylation-dependent increase in the association of hTRPC4 with Na(+)/H(+) exchanger regulatory factor. Furthermore, this translocation and association was defective upon mutation of Tyr-959 and Tyr-972 to phenylalanine. Significantly, inhibition of STKs was concomitant with a reduction in Ca(2+) influx in both native COS-7 cells and hTRPC4-expressing HEK293 cells, with cells expressing the Y959F/Y972F mutant exhibiting a reduced EGF response. These findings represent the first demonstration of a mechanism for phosphorylation to modulate TRPC channel function.     

表皮生长因子受体EGFR及其信号传导

表皮生长因子受体(epidermalgrowthfactorreceptor,EGFR)是ErbB家族成员之一,具有酪氨酸激酶活性,是一种重要的跨膜受体。EGFR被配体激活后启动胞内信号传导,经过细胞质中衔接蛋白、酶的级联反应,调节转录因子激活基因的转录,指导细胞迁移、黏附、增殖、分化、凋亡,且与肿瘤的形成和恶化密切相关。本文对EGFR的结构特性、几种重要的信号通路及各个信号通路之间的交联,以及信号的衰减等方面的研究进展进行了综述。     

Epidermal growth factor receptor signal transactivation

Cross-communication between heterologous signaling systems and the epidermal growth factor receptor (EGFR) has been shown to be critical for a variety of biological responses: EGFR transactivation when G-protein-coupled receptors (GPCRs) are stimulated represents the paradigm of an interreceptor network that is dependent on G-proteins, kinases, metalloproteases, and growth factor precursors. Investigating the mechanism of this process will help expand our knowledge of physiological regulatory mechanisms and diverse pathophysiological disorders.     

Epidermal growth factor stimulates phosphorylation of RAF-1 independently of receptor autophosphorylation and internalization.

Phosphorylation of the RAF-1 protooncogene product and activation of its associated serine/threonine kinase are common features of the response of cells to peptide growth factors. We have used wild-type and mutant epidermal growth factor (EGF) receptors to investigate mechanisms of RAF-1 phosphorylation. In vivo EGF treatment rapidly stimulated phosphorylation of RAF-1 exclusively on serine residues. Stimulation of RAF-1 phosphorylation occurred at 37 degrees C but not at 4 degrees C and persisted after dissociation of EGF from its receptor. EGF-induced RAF-1 serine phosphorylation required the intrinsic tyrosine kinase activity of the EGF receptor but was independent of EGF receptor self-phosphorylation and of ligand-induced receptor internalization. Down-regulation of protein kinase C did not affect the EGF-induced increase in RAF-1 phosphorylation. These data suggest that the activated tyrosine kinase activity of the EGF receptor enhances serine phosphorylation of RAF-1 via an intermediary molecule(s).     

Epidermal growth factor receptor tyrosine kinase phosphorylation of glucose-6-phosphate dehydrogenase in vitro

The specific tyrosine phosphorylation of glucose-6-phosphate dehydrogenase (G6PDH) by the epidermal growth factor (EGF) receptor in vitro is demonstrated. The Km values of the substrate G6PDH and of ATP for the receptor tyrosine kinase were ca. 1 and 10 microM, respectively. The rate of phosphorylation was EGF dependent, with a four-fold increase in Vmax in the presence of EGF. The phosphorylation was stimulated maximally by 0.2 microM or greater EGF, with an ED50 of ca. 20 nM which is consistent with the affinity of the solubilized receptor for EGF. Using conditions of 5 microM G6PDH, 100 microM ATP, 5 mM Mg2+, and 1 mM Mn2+, up to 0.3 mol phosphate was incorporated into 1 mol of the 55-kDa subunit of Baker's yeast G6PDH. Tryptic peptide mapping revealed several unique phosphopeptides for both Baker's yeast and bovine adrenal G6PDH. The patterns of phosphopeptides for a given enzyme were identical for basal and EGF-stimulated phosphorylation.     

Epidermal growth factor: relationship between receptor down regulation in cultured NRK cells and epidermal growth factor enhancement of phosphorylation of a 170000 molecular weight membrane protein in vitro

Incubation of confluent nondividing NRK cells in serum-free media with unlabeled epidermal growth factor (EGF) leads to a reduction in the specific binding capacity for 125I-labeled EGF. This modulation of the binding capacity for 125I-labeled EGF by unlabeled EGF, termed receptor down regulation, was dependent on EGF concentration and time. Membranes from untreated NRK cells have a phosphorylating system which catalyzed in vitro the phosphorylation of numerous membrane components; this phosphorylating system was stimulated by EGF. Although EGF enhanced the phosphorylation of many membrane proteins, one major component with Mr 170K and a minor band of Mr 150K were primarily affected. A comparison of the membrane phosphoproteins of untreated and down-regulated cells by in vitro phosphorylation and NaDodSO4 gel electrophoresis revealed that down regulation of EGF receptors results in a specific decrease in 32P phosphorylation of the 170K- and 150K-dalton components to subsequent stimulation with EGF in vitro. We further characterized the modulation of phosphorylation of the 170K protein by down regulation with EGF and found it to be dependent on EGF concentration and time. These studies demonstrated a correlation between the loss of 125I-labeled EGF binding activity by the cells and the loss of the vitro EGF-dependent 32P phosphorylation of the 170K-dalton membrane protein. In addition, the results suggest that the major 170K Mr phosphoprotein band is a component of the receptor for EGF which is a substrate of the phosphorylation reaction.     

Epidermal growth factor receptor: mechanisms of activation and signalling

The epidermal growth factor (EGF) receptor (EGFR) is one of four homologous transmembrane proteins that mediate the actions of a family of growth factors including EGF, transforming growth factor-alpha, and the neuregulins. We review the structure and function of the EGFR, from ligand binding to the initiation of intracellular signalling pathways that lead to changes in the biochemical state of the cell. The recent crystal structures of different domains from several members of the EGFR family have challenged our concepts of these processes.     

Epidermal growth factor receptor: Elements of intracellular communication

While EGF has an important function in cell growth regulation, the molecular mechanisms by which intracellular signal connect the EGF: receptor complex on the plasma membrane with the initiation of DNA synthesis and mitogenesis is not well understood. The discovery that rasGAP, PI-3 kinase and PLC-gamma 1 are substrates for the EGF receptor tyrosine kinase has provided a beginning in understanding the biochemistry underlying growth factor receptor transduction.     

Epidermal growth factor receptor: its role in Drosophila eye differentiation and cell survival

The Drosophila epidermal growth factor receptor (EGFR), functioning through the Ras/Raf/MAPK pathway, promotes cell proliferation and differentiation. Recent work has demonstrated that EGFR functions via the same Ras/Raf/MAPK pathway to promote cell survival. This review summarizes the role of EGFR in differentiation and survival during Drosophila eye development.     

Epidermal growth factor receptors: function modulation by phosphorylation and glycosylation interplay

Post-translational modifications (PTMs) of proteins induce structural and functional changes that are most often transitory and difficult to follow and investigate in vivo. In silico prediction procedures for PTMs are very valuable to foresee and define such transitory changes responsible for the multifunctionality of proteins. Epidermal growth factor receptor (EGFR) is such a multifunctional transmembrane protein with intrinsic tyrosine kinase activity that is regulated primarily by ligand-stimulated transphosphorylation of dimerized receptors. In human EGFR, potential phosphorylation sites on Ser, Thr and Tyr residues including five autophosphorylation sites on Tyr were investigated using in silico procedures. In addition to phosphorylation, O-GlcNAc modifications and interplay between these two modifications was also predicted. The interplay of phosphorylation and O-GlcNAc modification on same or neighboring Ser/Thr residues is termed as Yin Yang hypothesis and the interplay sites are named as Yin Yang sites. Amongst these modification sites, one residue is localized in the juxtamembrane (Thr 654) and two are found in the catalytic domain (Ser 1046/1047) of the EGFR. We propose that, when EGFR is O-GlcNAc modified on Thr 654, EGFR may be transferred from early to late endosomes, whereas when EGFR is O-GlcNAc modified on Ser 1046/1047 desensitization of the receptor may be prevented. These findings suggest a complex interplay between phosphorylation and O-GlcNAc modification resulting in modulation of EGFR's functionality.     

Growth factor receptor signalling: location, location, location

Ligand binding to plasma membrane receptors initiates a series of events culminating in a variety of changes in cellular phenotypes. Although numerous publications have documented the activation/inactivation of signalling molecules following receptor binding, relatively few investigations have focused on the cellular compartment responsible for either initiating or selecting the particular pathway that mediates the response. Specifically, does receptor signalling occur only at the plasma membrane; is signalling dependent upon the location of defined endosome populations; or are components of both plasma membrane and endosomal activity operative depending upon the particular signalling pathway or cell type? This review addresses aspects of these questions by discussing the evidence supporting or contrasting the interplay between the endocytic and signalling systems for a subset of tyrosine kinase, serine/threonine kinase and G-protein-coupled receptors.     

Epidermal growth factor-stimulated tyrosine phosphorylation of caveolin-1. Enhanced caveolin-1 tyrosine phosphorylation following aberrant epidermal growth factor receptor status

Caveolin-1 is the major coat protein of caveolae and has been reported to interact with various intracellular signaling molecules including the epidermal growth factor (EGF) receptor. To investigate the involvement of caveolin-1 in EGF receptor action, we used mouse B82L fibroblasts transfected with (a) wild type EGF receptor, (b) a C-terminally truncated EGF receptor at residue 1022, (c) a C-terminally truncated EGF receptor at residue 973, or (d) a kinase-inactive EGF receptor (K721M). Following EGF treatment, there was a distinct electrophoretic mobility shift of the caveolin-1 present in cells expressing the truncated forms of the EGF receptor, but this shift was not detectable in cells bearing either normal levels of the wild type EGF receptor or a kinase-inactive receptor. This mobility shift was also not observed following the addition of other cell stimuli, such as platelet-derived growth factor, insulin, basic fibroblast growth factor, or phorbol 12-myristate 13-acetate. Analysis of caveolin-1 immunoprecipitates from EGF-stimulated or nonstimulated cells demonstrated that the EGF-induced mobility shift of caveolin-1 was associated with its tyrosine phosphorylation in cells expressing truncated EGF receptors. Maximal caveolin-1 phosphorylation was achieved within 5 min after exposure to 10 nM EGF and remained elevated for at least 2 h. Additionally, several distinct phosphotyrosine-containing proteins (60, 45, 29, 24, and 20 kDa) were co-immunoprecipitated with caveolin-1 in an EGF-dependent manner. Furthermore, the Src family kinase inhibitor, PP1, does not affect autophosphorylation of the receptor, but it does inhibit the EGF-induced mobility shift and phosphorylation of caveolin-1. Conversely, the MEK inhibitors PD98059 and UO126 could attenuate EGF-induced mitogen-activated protein kinase activation, they do not affect the EGF-induced mobility shift of caveolin-1. Because truncation and overexpression of the EGF receptor have been linked to cell transformation, these results provide the first evidence that the tyrosine phosphorylation of caveolin-1 occurs via an EGF-sensitive signaling pathway that can be potentiated by an aberrant activity or expression of various forms of the EGF receptor.     

Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor

Epidermal growth factor (EGF) receptor from A-431 cells was purified by affinity chromatography with monoclonal anti-receptor antibodies. The purified radiolabeled receptor was incubated with EGF and then analyzed by gel electrophoresis under nondenaturing conditions. In these gels, the EGF receptor migrates in two forms: a fast-migrating (low) form and an EGF-induced slow-migrating (high) form. On the basis of the various control and calibration experiments described, it is concluded that the low form represents the monomeric 170-kilodalton EGF receptor and the high form represents an EGF receptor dimer. The binding of EGF causes a rapid, temperature-sensitive dimerization of the EGF receptor. Receptor dimerization is fully reversible and involves saturable, noncovalent interactions that are stable at neutral pH and in nonionic detergents. Both the monomeric and dimeric forms of the receptor bind EGF and undergo self-phosphorylation. The dimeric form of the receptor may possess higher ligand binding affinity, and it seems to be phosphorylated earlier than the monomeric form following the addition of EGF and [gamma-32P]ATP. On the basis of these results, it is concluded that receptor oligomerization is an intrinsic property of the occupied EGF receptor and that it may play a role in the activation of the kinase function and the subsequent transmembrane signaling process.     

Epidermal growth factor-induced truncation of the epidermal growth factor receptor

NIH-3T3 cells expressing the human epidermal growth factor (EGF) receptor were used in experiments to determine the fate of the EGF receptor in cells continuously exposed to EGF. EGF receptor was immunoprecipitated from cells labeled for 12 h with [35S] methionine in the absence or presence of 10 nM EGF. As expected, a single Mr = 170,000 polypeptide representing the mature EGF receptor was immune-precipitated from control cells. Surprisingly, immune precipitates from EGF-treated cells contained a prominent Mr = 125,000 receptor species, in addition to the Mr = 170,000 mature receptor. The Mr = 125,000 species was shown to be derived from the Mr = 170,000 form by pulse-chase experiments, in which the Mr = 170,000 receptor chased into the Mr = 125,000 form when EGF was included during the chase and by partial proteolysis. Both proteins became extensively phosphorylated on tyrosine residues in immune precipitate kinase assays. Treatment of immune precipitates with endoglycosidase F changed the apparent molecular weight of the Mr = 170,000 receptor to Mr = 130,000 and of the Mr = 125,000 form to Mr = 105,000, indicating that the appearance of the Mr = 125,000 protein was probably due to proteolysis. Antibody against the carboxyl terminus of the mature EGF receptor recognized the Mr = 125,000 protein, whereas antibody against the amino terminus did not. Incubation of cells with leupeptin prior to and during EGF addition inhibited processing to the Mr = 125,000 species. Methylamine and low temperature also inhibited the EGF-induced processing to the Mr = 125,000 form. These data suggest a possible role for proteolysis of the EGF receptor in receptor function.