Mechanisms of vascular angiotensin II surface receptor regulation by epidermal growth factor

We investigated mechanisms by which epidermal growth factor (EGF) reduces angiotensin II (AngII) surface receptor density and stimulated actions in vascular smooth muscle cells (VSMC). EGF downregulated specific AngII radioligand binding in intact cultured rat aortic smooth muscle cells but not in cell membranes and also inhibited AngII-stimulated contractions of aortic segments. Inhibitors of cAMP-dependent kinases, PI-3 kinase, MAP kinase, cyclooxygenase, and calmodulin did not prevent EGF-mediated downregulation of AngII receptor binding, whereas the EGF receptor kinase inhibitor AG1478 did. Total cell AngII AT1a receptor protein content of EGF-treated and untreated cells, measured by immunoblotting, did not differ. Actinomycin D or cytochalasin D, which interacts with the cytoskeleton, but not the protein synthesis inhibitor cycloheximide, prevented EGF from downregulating AngII receptor binding. Consistently, EGF inhibited AngII-stimulated formation of inositol phosphates in the presence of cycloheximide but not in the presence of actinomycin D or cytochalasin D. In conclusion, EGF needs an intact signal transduction pathway to downregulate AngII surface receptor binding, possibly by altering cellular location of the receptors.     

Mechanisms for oncogenic activation of the epidermal growth factor receptor

The Epidermal growth factor receptor (EGFR) is a membrane spanning glycoprotein, which frequently has been implicated in various cancer types. The mechanisms by which EGFR becomes oncogenic are numerous and are often specific for each cancer type. In some tumors, EGFR is activated by autocrine/paracrine growth factor loops, whereas in others activating mutations promote EGFR signaling. Overexpression and/or amplification of the EGFR gene are prevalent in many cancer types leading to aberrant EGFR signaling. In addition, failure to attenuate receptor signaling by receptor downregulation can also lead to cellular transformation. Heterodimerization of EGFR with ErbB2 inhibits downregulation of EGFR and thereby prolongs growth factor signaling. This also indicates that cross-talk between EGFR and heterologous receptor systems serves as another mechanism for oncogenic activation of EGFR. Because of its role in tumor promotion, the EGFR has been intensely studied as a therapeutic target. There are currently two major mechanisms by which the EGFR is targeted: antibodies binding to the extracellular domain of EGFR and small-molecule tyrosine-kinase inhibitors. However, tumorigenesis is a multi-step process involving several mutations, which might explain why EGFR therapeutics has only been partially successful. This highlights the importance of pinpointing the mechanisms by which EGFR becomes oncogenic in a particular cancer. In this review, each of the above mentioned mechanisms will be discussed, as a detailed molecular and genetic understanding of how EGFR contributes to the malignant phenotype might offer new promise for the design, development and clinical evaluation of future tumor-specific anticancer approaches.     

Estrogen regulation of epidermal growth factor receptor messenger ribonucleic acid

Previous studies have demonstrated that 17 beta-estradiol (E2) causes a 3-fold increase in epidermal growth factor (EGF) receptors in uterine membranes. We now report that the increase in uterine EGF receptor levels is due to an increase in the steady-state levels of EGF receptor mRNA. After a single E2 injection, EGF receptor mRNA levels, as determined by RNA blots, increase 3- to 4-fold between 1 and 3 h, remain elevated at 6 h, and decline between 12 and 18 h. The effect is specific for E2 since the nonestrogenic hormones progesterone, dexamethasone, 5 alpha-dihydrotestosterone, and the inactive stereoisomer of E2, 17 alpha-estradiol, are without effect. E2-Mediated increases in EGF receptor mRNA levels are blocked by actinomycin D but not by puromycin. Taken together, these results indicate that E2 regulates the level of EGF receptor by increasing the steady-state concentration of EGF receptor mRNA in vivo.     

Intrapeptide autophosphorylation of the epidermal growth factor receptor: regulation of kinase catalytic function by receptor dimerization

The epidermal growth factor (EGF) receptor is a transmembrane polypeptide of 170 000 daltons (Da) with a cytoplasmically facing protein kinase domain. The regulation of the tyrosine kinase activity of the EGF receptor by added EGF and by receptor association state was studied in an in vitro system. The rate of autophosphorylation of the solubilized and purified EGF receptor was found to be independent of receptor concentration. To determine whether the zero-order kinetics observed point to intrapeptide phosphorylation, we measured the sedimentation characteristics of the undenatured solubilized receptor. The receptor was found to exist in two association-dissociation states-a monomeric 7.7S form and a dimeric 12S form. The 7.7S form is an active tyrosine kinase; it has high basal activity, and the activity is not further stimulated by EGF; it appears to be an EGF-independent form of the receptor kinase. The 12S form is devoid of catalytic activity, but in the presence of EGF it dissociates into the active monomeric form. Freshly purified receptor preparations contain mainly the monomeric receptor, have high basal kinase activity, and show low EGF stimulatability (less than 1.3-fold). Aging of the receptor results in progressive dimerization and decay of EGF-independent kinase activity (and increase in EGF stimulatability). All of these processes are reversed in the presence of EGF or dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)     

SOCS2 induces neurite outgrowth by regulation of epidermal growth factor receptor activation

Suppressor of cytokine signaling (SOCS) 2 is a negative regulator of growth hormone (GH) signaling that regulates body growth postnatally and neuronal differentiation during development. SOCS2 binds to the GH receptor and inhibits GH signaling, including attenuation of STAT5 activation. Here we describe a new function and mechanism of action for SOCS2. Overexpression of SOCS2 in central nervous system neurons promoted neurite outgrowth, and in PC12 cells, neurite outgrowth was induced under nondifferentiating conditions, leading to inhibition of the neurite-inhibitory GTPase Rho and activation of the neurite-promoting GTPase Rac1. Addition of the epidermal growth factor receptor (EGFR) inhibitors PP3 or AG490 or the Src kinase inhibitor PP2 blocked the SOCS2-induced neurite outgrowth. The overexpressed SOCS2 bound to the EGFR, which was constitutively phosphorylated at Tyr845, the Src binding site. Overexpression of the phosphatase SHP-2 reduced the constitutive EGFR phosphorylation and subsequent neurite outgrowth. SOCS2 expression also resulted in a modest 30% decrease in phosphorylation of STAT5b at Tyr699, which is the primary site on STAT5 phosphorylated by GH; however, total tyrosine phosphorylation of STAT5 was decreased by 75-80% under basal and epidermal growth factor-stimulated conditions. Our findings suggest that SOCS2 regulates EGFR phosphorylation, leading to regulation of neurite outgrowth through a novel pathway that is distinct from GH.     

An insertional mutant of epidermal growth factor receptor allows dissection of diverse receptor functions

[This corrects the article on p. 2669 in vol. 6, PMID: 2824188.].     

Phosphorylation of the platelet-derived growth factor receptor-beta and epidermal growth factor receptor by G protein-coupled receptor kinase-2. Mechanisms for selectivity of desensitization

Accumulating evidence suggests that receptor protein-tyrosine kinases, like the platelet-derived growth factor receptor-beta (PDGFRbeta) and epidermal growth factor receptor (EGFR), may be desensitized by serine/threonine kinases. One such kinase, G protein-coupled receptor kinase-2 (GRK2), is known to mediate agonist-dependent phosphorylation and desensitization of multiple heptahelical receptors. In testing whether GRK2 could phosphorylate and desensitize the PDGFRbeta, we first found by phosphoamino acid analysis that cells expressing GRK2 could serine-phosphorylate the PDGFRbeta in an agonist-dependent manner. Augmentation or inhibition of GRK2 activity in cells, respectively, reduced or enhanced tyrosine phosphorylation of the PDGFRbeta but not the EGFR. Either overexpressed in cells or as a purified protein, GRK2 demonstrated agonist-promoted serine phosphorylation of the PDGFRbeta and, unexpectedly, the EGFR as well. Because GRK2 did not phosphorylate a kinase-dead (K634R) PDGFRbeta mutant, GRK2-mediated PDGFRbeta phosphorylation required receptor tyrosine kinase activity, as does PDGFRbeta ubiquitination. Agonist-induced ubiquitination of the PDGFRbeta, but not the EGFR, was enhanced in cells overexpressing GRK2. Nevertheless, GRK2 overexpression did not augment PDGFRbeta down-regulation. Like the vast majority of GRK2 substrates, the PDGFRbeta, but not the EGFR, activated heterotrimeric G proteins allosterically in membranes from cells expressing physiologic protein levels. We conclude that GRK2 can phosphorylate and desensitize the PDGFRbeta, perhaps through mechanisms related to receptor ubiquitination. Specificity of GRK2 for receptor protein-tyrosine kinases, expressed at physiologic levels, may be determined by the ability of these receptors to activate heterotrimeric G proteins, among other factors.     

Modulation of the epidermal growth factor receptor by basic fibroblast growth factor

Treatment of Swiss 3T3 fibroblasts with basic fibroblast growth factor (bFGF) lead to a rapid reduction in epidermal growth factor (EGF) binding and a slower inhibition of EGF receptor autophosphorylation. The reduction in binding was due to a complete loss of the highest affinity EGF binding sites and a reduction in the lower affinity binding sites. Neither the inhibition of EGF binding nor the inhibition of EGF receptor autophosphorylation required protein kinase C. Treatment of cells with bFGF stimulated the phosphorylation of the EGF receptor, which persisted for several hours. The inhibition of EGF receptor autophosphorylation by bFGF was reduced in the presence of cycloheximide. However, cycloheximide had no effect on the reduction of EGF binding by bFGF. In contrast to these results with Swiss 3T3 fibroblasts, treatment of PC12 cells with bFGF lead to a reduction in EGF binding but no inhibition of EGF receptor autophosphorylation. Thus inhibited of EGF receptor autophosphorylation and inhibition of EGF binding can be uncoupled. © 1993 Wiley-Liss, Inc.     

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.     

Apical epidermal growth factor receptor signaling: regulation of stretch-dependent exocytosis in bladder umbrella cells

The apical surface of polarized epithelial cells receives input from mediators, growth factors, and mechanical stimuli. How these stimuli are coordinated to regulate complex cellular functions such as polarized membrane traffic is not understood. We analyzed the requirement for growth factor signaling and mechanical stimuli in umbrella cells, which line the mucosal surface of the bladder and dynamically insert and remove apical membrane in response to stretch. We observed that stretch-stimulated exocytosis required apical epidermal growth factor (EGF) receptor activation and that activation occurred in an autocrine manner downstream of heparin-binding EGF-like growth factor precursor cleavage. Long-term changes in apical exocytosis depended on protein synthesis, which occurred upon EGF receptor-dependent activation of mitogen-activated protein kinase signaling. Our results indicate a novel physiological role for the EGF receptor that couples upstream mechanical stimuli to downstream apical EGF receptor activation that may regulate apical surface area changes during bladder filling.     

Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor

Novel cancer chemotherapeutics are required to induce apoptosis by activating pro-apoptotic proteins. Both epidermal growth factor (EGF) and insulin-like growth factor (IGF) provide potent survival stimuli in many epithelia, and activation of their receptors is commonly observed in solid human tumors. Here we demonstrate that blockade of the EGF receptor by a new drug in phase III clinical trails for cancer, ZD1839, potently induces apoptosis in mammary epithelial cell lines and primary cultures, as well as in a primary pleural effusion from a breast cancer patient. We identified the mechanism of apoptosis induction by ZD1839. We showed that it prevents cell survival by activating the pro-apoptotic protein BAD. Moreover, we demonstrate that IGF transactivates the EGF receptor and that ZD1839 blocks IGF-mediated phosphorylation of MAPK and BAD. Many cancer therapies kill tumor cells by inducing apoptosis as a consequence of targeting DNA; however, the threshold at which apoptosis can be triggered through DNA damage is often different from that in normal cells. Our results indicate that by targeting a growth factor-mediated survival signaling pathway, BAD phosphorylation can be manipulated therapeutically to induce apoptosis.     

Spatial activation of ezrin by epidermal growth factor receptor and focal adhesion kinase co-ordinates epithelial cell migration

Epidermal growth factor receptor (EGFR) plays a critical role in the promotion of epithelial cell proliferation and migration. Previous studies have suggested a cooperative role between EGFR and integrin signalling pathways that enable efficient adhesion and migration but the mechanisms controlling this remain poorly defined. Here, we show that EGFR forms a complex with focal adhesion kinase in epithelial cells. Surprisingly, this complex enhances local Src activity at focal adhesions to promote phosphorylation of the cytoskeletal adaptor protein ezrin at Y478, leading to actomyosin contractility, suppression of focal adhesion dynamics and slower migration. We further demonstrate this regulation of Src is due to the suppression of PTP1B activity. Our data provide new insight into EGF-independent cooperation between EGFR and integrins and suggest transient interactions between these kinases at the leading edge of cells act to spatially control signalling to permit efficient motility.     

An insertional mutant of epidermal growth factor receptor allows dissection of diverse receptor functions.

Cultured NIH-3T3 cells devoid of endogenous EGF-receptors were transfected with cDNA constructs encoding normal human EGF-receptor and with a construct encoding an insertional mutant of the EGF-receptor containing four additional amino acids in the kinase domain after residue 708. Unlike the wild-type receptor expressed in these cells which exhibits EGF-stimulatable protein tyrosine kinase activity, the mutant receptor lacks protein tyrosine kinase activity both in vitro and in vivo. Despite this deficiency the mutant receptor is properly processed, it binds EGF and it exhibits both high and low affinity binding sites. Moreover, it undergoes efficient EGF-mediated endocytosis. However, EGF fails to stimulate DNA synthesis and is unable to stimulate the phosphorylation of S6 ribosomal protein in cells expressing this receptor mutant. Hence, it is proposed that the protein tyrosine kinase activity of EGF-receptor is essential for the initiation of S6 phosphorylation and for DNA synthesis induced by EGF. However, EGF-receptor processing, the expression of high and low affinity surface receptors and receptor internalization, require neither kinase activity nor receptor autophosphorylation. Interestingly, phorbol ester (TPA) fails to abolish the high affinity state and is also unable to stimulate the phosphorylation of this receptor mutant. This result is consistent with the notion that kinase-C phosphorylation of EGF-receptor is essential for the loss of high affinity EGF-receptors caused by TPA.     

Regulation of the epidermal growth factor receptor by phosphorylation

The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self-phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37 degrees C and exhibits a very low Km for ATP (0.2 microM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self-phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self-phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function.     

Molecular targeting in radiotherapy: epidermal growth factor receptor

Radiation therapy is utilized as a treatment to cure or manage cancer; however, because of risk to local healthy tissue-and a modest success rate of some radiotherapy-strategies have been sought that would increase the therapeutic index of the treatment while reducing damage to surrounding tissue. Cell and tissue irradiation stimulates a series of biochemical and molecular signals; various components of this ionizing radiation (IR)-inducible signal transduction cascade can promote the survival of tumor cells. Identification of interactions between IR and a signaling pathway creates an opportunity to target those signaling intermediates to improve the outcome of radiotherapy. The epidermal growth factor receptor (EGFR, also termed ErbB1) is involved in normal development and differentiation of epithelial cells as well as in tumorigenesis. The EGFR is activated by IR, thus making this receptor and other members of the ErbB family important targets for radiosensitizing molecular interventions. Recent approaches have utilized monoclonal antibodies, small molecules, and transgenic technologies to undermine the kinase activity of EGFR.