Proprotein convertase PC7 enhances the activation of the EGF receptor pathway through processing of the EGF precursor

Although the processing profile of the membrane-bound epidermal growth factor precursor (pro-EGF) is tissue-specific, it has not been investigated at the cellular level nor have the cognate proteinases been defined. Among the proprotein convertases (PCs), only the membrane-bound PC7, the most ancient and conserved basic amino acid-specific PC family member, induces the processing of pro-EGF into an ～115-kDa transmembrane form (EGF-115) at an unusual VHPR(290)↓A motif. Because site-directed mutagenesis revealed that Arg(290) is not critical, the generation of EGF-115 by PC7 is likely indirect. This was confirmed by testing a wide range of protease inhibitors, which revealed that the production of EGF-115 is most probably achieved via the activation by PC7 of a latent serine and/or cysteine protease(s). EGF-115 is more abundant at the cell surface than pro-EGF and is associated with a stronger EGF receptor (EGFR) activation, as evidenced by higher levels of phosphorylated ERK1/2. This suggests that the generation of EGF-115 represents a regulatory mechanism of juxtacrine EGFR activation. Thus, PC7 is distinct from the other PCs in its ability to enhance the activation of the cell surface EGFR.     

c-Src trafficking and co-localization with the EGF receptor promotes EGF ligand-independent EGF receptor activation and signaling

c-Src is a non-receptor tyrosine kinase that associates with both the plasma membrane and endosomal compartments. In many human cancers, especially breast cancer, c-Src and the EGF receptor (EGFR) are overexpressed. Dual overexpression of c-Src and EGFR correlates with a Src-dependent increase in activation of EGFR, and synergism between these two tyrosine kinases increases the mitogenic activity of EGFR. Despite extensive studies of the functional interaction between c-Src and EGFR, little is known about the interactions in the trafficking pathways for the two proteins and how that influences signaling. Given the synergism between c-Src and EGFR, and the finding that EGFR is internalized and can signal from endosomes, we hypothesized that c-Src and EGFR traffic together through the endocytic pathway. Here we use a regulatable c-SrcGFP fusion protein that is a bona fide marker for c-Src to show that c-Src undergoes constitutive macropinocytosis from the plasma membrane into endocytic compartments. The movement of c-Src was dependent on its tyrosine kinase activity. Stimulation of cells with EGF revealed that c-Src traffics into the cell with activated EGFR and that c-Src expression and kinase activity prolongs EGFR activation. Surprisingly, even in the absence of EGF addition, c-Src expression induced activation of EGFR and of EGFR-mediated downstream signaling targets ERK and Shc. These data suggest that the synergy between c-Src and EGFR also occurs as these two kinases traffic together, and that their co-localization promotes EGFR-mediated signaling.     

Epidermal growth factor (EGF) stimulates EGF receptor synthesis

Epidermal growth factor (EGF) binds to the extracellular domain of a specific 170,000-dalton transmembrane glycoprotein; this results in rapid removal of both ligand and receptor from the cell surface. In WB cells, a rat hepatic epithelial cell line, ligand-directed receptor internalization leads to receptor degradation. We tested whether the EGF receptor was replenished at a constitutive or enhanced rate following EGF binding by immunoprecipitating biosynthetically labeled EGF receptor from cells cultured with [35S]methionine. EGF stimulated receptor synthesis within 2 h in a dose-dependent manner; this was particularly evident when examining the nascent form of the receptor. To determine the site of EGF action, total WB cell RNA was transferred to nitrocellulose paper after electrophoresis and was hybridized to cDNA probes from both the external and cytoplasmic coding regions of the human EGF receptor. EGF increased receptor mRNA by 3-5-fold. Therefore, at least in some cells, the surface action of EGF that leads to EGF receptor degradation is counterbalanced by a positive effect on receptor synthesis.     

EGF receptor.

The receptor for the epidermal growth factor (EGF) and related ligands (EGFR), the prototypal member of the superfamily of receptors with intrinsic tyrosine kinase activity, is widely expressed on many cell types, including epithelial and mesenchymal lineages. Upon activation by at least five genetically distinct ligands (including EGF, transforming growth factor-alpha (TGF alpha) and heparin-binding EGF (HB-EGF)), the intrinsic kinase is activated and EGFR tyrosyl-phosphorylates itself and numerous intermediary effector molecules, including closely-related c-erbB receptor family members. This initiates myriad signaling pathways, some of which attenuate receptor signaling. The integrated biological responses to EGFR signaling are pleiotropic including mitogenesis or apoptosis, enhanced cell motility, protein secretion, and differentiation or dedifferentiation. In addition to being implicated in organ morphogenesis, maintenance and repair, upregulated EGFR signaling has been correlated in a wide variety of tumors with progression to invasion and metastasis. Thus, EGFR and its downstream signaling molecules' are targets for therapeutic interventions in wound repair and cancer.     

The TGF-alpha precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction

The 50 amino acid form of TGF-alpha is cleaved from a conserved integral membrane glycoprotein by a protease that, in many tumor cells, appears to be limiting. To test whether the membrane-bound precursor has biological activity in the absence of processing, we introduced amino acid substitutions at the proteolytic cleavage sites. BHK cells transfected with expression vectors containing these altered sequences do not secrete detectable levels of mature TGF-alpha into the medium, but express high levels of proTGF-alpha at the cell surface. Coincubation of these BHK cells with A431 cells demonstrates that membrane-bound proTGF-alpha may bind to EGF receptors on the surface of contiguous cells, induce receptor autophosphorylation, and thereby produce a rapid rise in A431 intracellular calcium levels. Thus, proTGF-alpha can be biologically active in the absence of processing, a fact that may have implications for the integral membrane precursors of related growth factors.     

Serum proteome mapping of EGF transgenic mice reveal mechanistic biomarkers of lung cancer precursor lesions with clinical significance for human adenocarcinomas

Atypical adenomatous hyperplasia (AAH) of the lung is a pre-invasive lesion (PL) with high risk of progression to lung cancer (LC). However, the pathways involved are uncertain. We searched for novel mechanistic biomarkers of AAH in an EGF transgenic disease model of lung cancer. Disease regulated proteins were validated by Western immunoblotting and immunohistochemistry (IHC) of control and morphologically altered respiratory epithelium. Translational work involved clinical resection material. Collectively, 68 unique serum proteins were identified by 2DE-MALDI-TOF mass spectrometry and 13 reached statistical significance (p?<?0.05). EGF, amphiregulin and the EGFR endosomal sorting protein VPS28 were induced up to 5-fold while IHC confirmed strong induction of these proteins. Furthermore, ApoA1, α-2-macroglobulin, and vitamin-D binding protein were nearly 6- and 2-fold upregulated in AAH; however, ApoA1 was oppositely regulated in LC to evidence disease stage dependent regulation of this tumour suppressor. Conversely, plasminogen and transthyretin were highly significantly repressed by 3- and 20-fold. IHC confirmed induced ApoA1, Fetuin-B and transthyretin expression to influence calcification, inflammation and tumour-infiltrating macrophages. Moreover, serum ApoA4, ApoH and ApoM were 2-, 2- and 6-fold repressed; however tissue ApoM and sphingosine-1-phosphate receptor expression was markedly induced to suggest a critical role of sphingosine-1-phosphate signalling in PL and malignant transformation. Finally, a comparison of three different LC models revealed common and unique serum biomarkers mechanistically linked to EGFR, cMyc and cRaf signalling. Their validation by IHC on clinical resection material established relevance for distinct human lung pathologies. In conclusion, we identified mechanistic biomarker candidates recommended for in-depth clinical evaluation.     

Epidermal growth factor (EGF) in the goat uterus: immunohistochemical localization of EGF and EGF receptor and effect of EGF on uterine activity in vivo

This study examined the distribution of immunoreactive epidermal growth factor (EGF) and EGF receptor (EGF-R) in the uterus and the effects of EGF on uterine activity in goats. Immunohistochemistry of EGF and EGF-R in the uteri showed distinct staining in the luminal and glandular epithelium and slight to moderate staining in the stromal and myometrial cells. To examine possible roles of the EGF system in the regulation of uterine activity, pressure changes in the intrauterine balloon were determined after intraluminal infusion of EGF into the uterine horn. Either at estrus or diestrus (9 to 14 days after the first day of estrus), treatment with 1 or 5 microg of EGF gradually reduced uterine activity, whereas infusion of the vehicle alone had no effect. The maximum reduction in uterine activity was seen 4 h after the treatment with 1 microg of EGF (40% to 45% reduction in the area surrounded by the contraction curve and its baseline), and the activity slowly returned thereafter. These results suggest that EGF in the uterus may play a role in regulating uterine activity in goats.     

Cytotoxic properties of DAB486EGF and DAB389EGF, epidermal growth factor (EGF) receptor-targeted fusion toxins

Elevated expression of the receptor for epidermal growth factor (EGF) is a characteristic of several malignancies including those of the breast, bladder, prostate, lung, and neuroglia. To therapeutically target the cytotoxic action of diphtheria toxin to EGF receptor-expressing tumor cells, we have constructed a hybrid gene in which the sequences for the binding domain of diphtheria toxin have been replaced by those for human EGF. The resulting fusion toxins, DAB486EGF and DAB389EGF, bind specifically to the EGF receptor and inhibit protein synthesis in a variety of EGF receptor expressing human tumor cell lines with an IC50 as low as 0.1 pM. Comparisons of DAB486EGF and DAB389EGF showed that DAB389EGF was consistently 10- to 100-fold more cytotoxic than DAB486EGF. Like diphtheria toxin, the cytotoxic action of DAB389EGF results from ADP-ribosylation of elongation factor-2 and is sensitive to the action of chloroquine. Studies of the kinetics of cellular intoxication showed that a 15-min exposure of EGF receptor-expressing A431 cells to DAB389EGF results in complete protein synthesis inhibition within 4 h. Furthermore, inhibition of protein synthesis results in elimination of human tumor cell colonies. These findings show that DAB389EGF is a potential therapeutic agent for a wide variety of EGF receptor-expressing solid tumors.     

Effect of EGF administration on EGF receptor mRNA in hCG producing tumor

In this study we examined the expression of EGF receptor mRNA after EGF administration in hCG producing tumor (choriocarcinoma). We transplanted the tissue of choriocarcinoma into female nude mice and investigated the effects of EGF on the growth of tumors, the binding activity of EGF receptor and the expression of EGF receptor mRNA in the tumor tissues. Two doses of EGF 5.0 micrograms, 50 micrograms and phosphate buffered saline as a control were injected subcutaneously every day for four weeks. Removed tumors were used for immunocytochemical studies and EGF receptor mRNA investigations. HCG and EGF receptors were detected immunocytochemically in the tumor. The low dose EGF employed stimulated the tumor growth while the high dose EGF inhibited the tumor growth compared with that of the control group. The binding activity of EGF receptor and the expression of EGF receptor mRNA also changed in accordance with the stimulation or inhibition of tumor growth. The growth of hCG producing tumor by EGF administration appeared to be dependent upon the binding activity of EGF receptor and the expression of EGF receptor mRNA.     

EGF family ligand-dependent phenotypic modulation of smooth muscle cells through EGF receptor

The phenotypic modulation of smooth muscle cells (SMCs) is closely associated with the development and progression of various SMC diseases. We investigated the molecular mechanism of phenotypic modulation triggered by EGF family ligands using a primary culture system of differentiated SMCs. Among four EGF-receptor (EGFR) family members, the EGFR was solely activated by EGF, heparin-binding EGF (HB-EGF), transforming growth factor alpha (TGF alpha), epiregulin (ER), and betacellulin (BTC), resulting in induction of phenotypic modulation of SMCs. This effect was mediated through the coordinated activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) pathways. These results suggest that EGF family ligand- and EGFR-triggered signaling pathways are critically involved in the phenotypic modulation of SMCs.     

EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate

EGF-R [EGF (epidermal growth factor) receptor] ligands can promote or inhibit cell growth. The biological outcome of receptor activation is dictated, at least in part, by ligand-specified patterns of endocytic trafficking. EGF-R trafficking downstream of the ligands EGF and TGF-alpha (transforming growth factor-alpha) has been investigated extensively. However, less is known about EGF-R fates induced by the ligands BTC (betacellulin) and AR (amphiregulin). We undertook comparative analyses to identify ligand-specific molecular events that regulate EGF-R trafficking and degradation. EGF (17 nM) and BTC (8.5 nM) induced significant EGF-R degradation, with or without ectopic expression of the ubiquitin ligase Cbl. Human recombinant AR (17 nM) failed to affect receptor degradation in either case. Notably, levels of ligand-induced EGF-R ubiquitination did not correlate strictly with receptor degradation. Dose-response experiments revealed that AR at a saturating concentration was a partial agonist at the EGF-R, with approx. 40% efficacy (relative to EGF) at inducing receptor tyrosine phosphorylation, ubiquitination and association with Cbl. EGF-R down-regulation and degradation also were compromised upon cell stimulation with AR (136 nM). These outcomes correlated with decreased degradation of the Cbl substrate and internalization inhibitor hSprouty2. Downstream of the hSprouty2 checkpoint in AR-stimulated cells, Cbl-free EGF-R was incorporated into endosomes from which Cbl-EGF-R complexes were excluded. Our results suggest that the AR-specific EGF-R fate results from decreased hSprouty2 degradation and reduced Cbl recruitment to underphosphorylated EGF-R, two effects that impair EGF-R trafficking to lysosomes.     

Epidermal growth factor (EGF)-stimulated tyrosine phosphorylation and EGF receptor degradation in cells expressing EGF receptors truncated at residue 973.

Epidermal growth factor (EGF)-stimulated tyrosine phosphorylation of proteins was examined in cells expressing wild-type (WT-EGFR) EGF receptors or EGF receptors truncated at residue 973 (973-EGFR). A much broader spectrum of tyrosine phosphorylated proteins was found following EGF treatment of 973-EGFR expressing cells compared with cells expressing wild-type receptors. Several additional ras GTPase activating protein-associated tyrosine phosphorylated proteins were found in EGF-treated 973-EGFR cells relative to WT-EGFR cells. Additional tyrosine-phosphorylated proteins were also found to co-immunoprecipitate with phospholipase C gamma 1 (PLC gamma 1) following EGF treatment of cells expressing 973-EGFR relative to cells expressing WT-EGFR. EGF-stimulated tyrosine phosphorylation of PLC gamma 1 was found in cells expressing WT-EGFR, but not in cells expressing 973-EGFR. WT-EGF receptor from EGF-treated cells bound well to bacterially expressed src homology (SH) regions of PLC gamma 1 and to a lesser extent to bacterially expressed GTPase activating protein SH regions. No binding of 973-EGF receptor to SH regions of either protein could be detected. EGF treatment greatly reduced the half-life of WT-EGFR, but had relatively little effect on the half-life of 973-EGFR. EGF induced internalization of 973-EGFR at a slower rate than WT-EGFR and caused the appearance of discrete receptor degradation products for both cell types. The data indicate that truncation of the EGF receptor at residue 973 alters receptor substrate specificity, decreases the rate of receptor internalization, and has an inhibitory effect on receptor degradation.