Localization Of Epidermal Growth Factor Receptor Research Articles

Cetuximab/C225-Induced Intracellular Trafficking of Epidermal Growth Factor Receptor

The monoclonal antibody C225 interacts with the ectodomain of the epidermal growth factor (EGF) receptor (EGFR) to block ligand binding and initiates receptor endocytosis and intracellular trafficking. The data herein show that C225-dependent EGFR trafficking relocalizes the receptor to the endoplasmic reticulum (ER) and nucleus. This mechanism, which also involves interaction of the C225-internalized receptor with the Sec61 translocon within the ER, is, in most respects, analogous to the pathway previously described for EGF-induced trafficking to the ER and nucleus. However, although inhibition of receptor tyrosine kinase activity blocks EGF-induced nuclear localization of the receptor, the same kinase inhibitors stimulate C225-dependent nuclear localization of EGFR in the nucleus. In contrast, the kinase inhibitor Lapatinib fails to stimulate nuclear accumulation of the receptor in C225-treated cells and does not provoke receptor dimerization as do inhibitors that recognize the open conformation of the receptor kinase. This suggests that inhibitor-dependent receptor dimerization may facilitate C225-induced receptor trafficking.

Prostaglandin E1 and misoprostol increase epidermal growth factor production in 3D-cultured human annulus cells

Background context Epidermal growth factor (EGF) is a peptide known to modulate a number of cellular responses including embryogenesis, cell proliferation, and cell survival. Little is known about EGF and its regulation in human annulus cells. Previous work has identified EGF and its receptor in control outer annulus disc tissue, but not in herniated tissue. Purpose To determine if human annulus cells express EGF in vitro, to determine if the epidermal growth factor-receptor (EGF-r) was expressed in vivo and in vitro in disc cells, to test the effect of EGF on annulus cell proliferation and proteoglycan production in vitro, and to test the effect of prostaglandin E1 (PGE1) and misoprostol on disc cell production of EGF in vitro. Study design/setting Studies were approved by the authors' Human Subjects Institutional review Board. Human disc tissue was used for immunocytochemistry, and human annulus cells were tested in vitro. Patient sample Thirty-four disc specimens were used for studies of proteoglycan production, cell proliferation, and EGF production in vitro. An additional nine discs were used for EGF-r immunolocalization. Methods Disc tissue was used for immunocytochemical studies for the localization of EGF-r and as a source for cultured annulus cells. Monolayer culture was used to test proliferation responses to 0, 25, 50, or 75 ng/mL EGF over a 2-day culture period. Three-dimensional (3D) culture in a collagen sponge was used to test 100,000 cells cultured in a paired experimental design over 14 days for production of EGF and proteoglycans. Cells were exposed to control conditions, or to either misoprostol at 8 ng/mL or PGE1 at 10 −7 M. Conditioned media was harvested and assayed using an enzyme-linked immunosorbent assay (ELISA) assay with the Human Protein Cytokine Antibody Array I kit. Replicate EGF relative intensity values were averaged and normalized to controls assayed on the same membrane. 3D-cultured cells were also used to confirm EGF gene expression using microarray analysis. Standard statistical methods were used to analyze results. Results Microarray analysis of mRNA from annulus cells in 3D culture confirmed expression of EGF, and immunocytochemistry verified the presence of EGF-r in vitro and in vivo. PGE1, at a dose of 10 −7 M, and misoprostol (a synthetic PGE1 analog) at a dose of 8 ng/mL, both significantly increased EGF levels in annulus cells cultured in 3D compared with control levels (p=.031 and .034, respectively). No significant difference, however, was seen in cell proliferation or in total sulfated proteoglycan production in EGF-exposed annulus cells. Conclusions Data showed that EGF is expressed and produced by annulus cells in vivo and in 3D culture, with significantly greater in vitro EGF produced in the presence of PGE1 or the PGE1 analog misoprostol. Misoprostol, developed for prevention/treatment of nonsteroidal anti-inflammatory-induced gastropathy, has now been reported to have some interesting anabolic effects stimulating osteoblasts during fracture healing and during ovariectomy in animal models. Exogenous EGF did not increase cell proliferation in monolayer, or total production of proteoglycans in 3D culture. Additional work is needed to further delineate the role of EGF in the human disc.

Quantum dot-based quantification revealed differences in subcellular localizationof EGFR and E-cadherin between EGFR-TKI sensitive and insensitive cancercells

Nanoparticle quantum dots (QDs) provide sharper and more photostable fluorescent signalsthan organic dyes, allowing quantification of multiple biomarkers simultaneously.In this study, we quantified the expression of epidermal growth factor receptor(EGFR) and E-cadherin (E-cad) in the same cells simultaneously by using secondaryantibody-conjugated QDs with two different emission wavelengths (QD605 and QD565)and compared the cellular distribution of EGFR and E-cad between EGFR-tyrosine kinaseinhibitor (TKI)-insensitive and -sensitive lung and head and neck cancer cell lines.Relocalization of EGFR and E-cad upon treatment with the EGFR-TKI erlotinib in thepresence of EGF was visualized and analyzed quantitatively. Our results showed thatQD-immunocytochemistry (ICC)-based technology can not only quantify basal levels ofmultiple biomarkers but also track the localization of the biomarkers upon biostimulation.With this new technology we found that in EGFR-TKI-insensitive cells, EGFRand E-cad were located mainly in the cytoplasm; while in sensitive cells, theywere found mainly on the cell membrane. After induction with EGF, both EGFRand E-cad internalized to the cytoplasm, but the internalization capability insensitive cells was greater than that in insensitive cells. Quantification also showedthat inhibition of EGF-induced EGFR and E-cad internalization by erlotinib inthe sensitive cells was stronger than that in the insensitive cells. These studiesdemonstrate substantial differences between EGFR-TKI-insensitive and -sensitivecancer cells in EGFR and E-cad expression and localization both at the basallevel and in response to EGF and erlotinib. QD-based analysis facilitates theunderstanding of the features of EGFR-TKI-insensitive versus -sensitive cancercells and may be used in the prediction of patient response to EGFR-targetedtherapy.

Antiproliferative effect of exemestane in lung cancer cells.

BackgroundRecent evidence suggests that estrogen signaling may be involved in the pathogenesis of non-small cell lung cancer (NSCLC). Aromatase is an enzyme complex that catalyses the final step in estrogen synthesis and is present in several tissues, including the lung. In the current study we investigated the activity of the aromatase inhibitor exemestane in human NSCLC cell lines H23 and A549.ResultsAromatase expression was detected in both cell lines. H23 cells showed lower protein and mRNA levels of aromatase, compared to A549 cells. Exemestane decreased cell proliferation and increased apoptosis in both cell lines, 48 h after its application, with A549 exhibiting higher sensitivity than H23 cells. Aromatase protein and mRNA levels were not affected by exemestane in A549 cells, whereas an increase in both protein and mRNA levels was observed in H23 cells, 48 h after exemestane application. Moreover, an increase in cAMP levels was found in both cell lines, 15 min after the administration of exemestane. In addition, we studied the effect of exemestane on epidermal growth factor receptor (EGFR) localization and activation. Exemestane increased EGFR activation 15 min after its application in H23 cells. Furthermore, we demonstrated a translocation of EGFR from cell membrane, 24 h after the addition of exemestane in H23 cells. No changes in EGFR activation or localization were observed in A549 cells.ConclusionOur findings suggest an antiproliferative effect of exemestane on NSCLC cell lines. Exemestane may be more effective in cells with higher aromatase levels. Further studies are needed to assess the activity of exemestane in NSCLC.

Altered EGFR localization and degradation in human breast cancer cells with an amphiregulin/EGFR autocrine loop

The epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AR) have been shown to be co-over expressed in breast cancer. We have previously shown that an AR/EGFR autocrine loop is required for SUM149 human breast cancer cell proliferation, motility and invasion. We also demonstrated that AR can induce these altered phenotypes when expressed in the normal mammary epithelial cell line MCF10A, or by exposure of these cells to AR in the medium. In the present studies, we demonstrate that SUM149 cells and immortalized human mammary epithelial MCF10A cells that over express AR (MCF10A AR) or are cultured in the presence of exogenous AR, express higher levels of EGFR protein than MCF10A cells cultured in EGF. Pulse–chase analysis showed that EGFR protein remained stable in the presence of AR, yet was degraded in the presence of EGF. Consistent with this observation, tyrosine 1045 on the EGFR, the c-cbl binding site, exhibited less phosphorylation following stimulation with AR than following stimulation with EGF. Ubiquitination of the receptor was also dramatically less following stimulation with AR than following stimulation with EGF. Flow cytometry analysis showed that EGFR remained on the cell surface following stimulation with AR but was rapidly internalized following stimulation with EGF. Immunofluorescence and confocal microscopy confirmed the flow cytometry results. EGFR in MCF10A cells cultured in the presence of EGF exhibited a predominantly intracellular, punctate localization. In stark contrast, SUM149 cells and MCF10A cells growing in the presence of AR expressed EGFR predominantly on the membrane and at cell–cell junctions. We propose that AR alters EGFR internalization and degradation in a way that favors accumulation of EGFR at the cell surface and ultimately leads to changes in EGFR signaling.

Internalization of EGF receptor following lipid rafts disruption in keratinocytes is delayed and dependent on p38 MAPK activation

The receptor for epidermal growth factor (EGF) plays an important role in epidermal keratinocytes and is known to move out of lipid raft after cholesterol depletion, leading to ligand-independent activation. Accumulation of evidence indicates the ability of EGF receptor (EGFR) to undergo internalization without participation of the ligand under the control of p38 MAPK during stress conditions. Since cholesterol depletion using methyl-beta-cyclodextrin is known to induce ligand-independent activation of EGFR in keratinocytes, we investigated by confocal microscopy and ligand-binding tests the processing and localization of EGFR following lipid raft disruption. Here, we report the dimerization and the slow internalization of the receptor accompanied by the delayed phosphorylation of tyrosine 1068 and its degradation by the proteasome. We also demonstrate the involvement of p38 MAPK during the process of internalization, which can be considered as a protective response to stress. Moreover, cholesterol-depleted keratinocytes recover their ability to proliferate during the recovery period that follows lipid raft disruption.

Distinctive E‐cadherin and epidermal growth factor receptor expression in metastatic and nonmetastatic head and neck squamous cell carcinoma

The authors investigated whether coexpression and localization of E-cadherin (E-cad) and epidermal growth factor receptor (EGFR) had predictive and/or prognostic correlations with lymph node metastasis and/or survival in patients with squamous cell carcinoma of the head and neck (SCCHN). Immunohistochemistry (IHC) of archival tissue was performed to measure expression of EGFR and E-cad in surgical specimens of SCCHN (n = 143) that included primary tumors (PTs) with positive lymph nodes (Tu+Met) and their paired lymph node metastases (LnMet), PTs with negative lymph nodes (Tu(-Met)), and benign tissue biopsies as normal controls. IHC staining was quantified as a weighted index and as the ratio of membrane to cytoplasmic staining. Correlative expression between EGFR and E-cad also was examined in SCCHN cell lines by immunoblotting and immunofluorescence analyses. Three distinct expression patterns of EGFR and E-cad were observed. Membrane localization of E-cad was significantly lower in the Tu+Met group than in the Tu(-Met) group (P = .01) and was associated inversely with lymph node status (P = .009). Wilcoxon analysis of the combined markers demonstrated that expression and/or membrane localization of EGFR and E-cad were correlated with disease-free survival and overall survival in patients with SCCHN. The study of SCCHN cell lines demonstrated that cells with positive but low EGFR expression and with negative E-cad expression were relatively resistant to the EGFR tyrosine kinase inhibitor erlotinib. The current study suggested that examining not only the expression but also the localization of EGFR and E-cad simultaneously may have clinical relevance in predicting lymph node metastasis, patient survival, and response to EGFR-targeted therapy in patients with SCCHN.

Mitochondrially localized EGFR is subjected to autophagic regulation and implicated in cell survival

Although generally acknowledged as a plasma membrane protein, the epidermal growth factor (EGF) receptor has been found in the nucleus and subcellular organelles. Recently, the mitochondrial localization of the EGF receptor (EGFR) was reported; nevertheless, the molecular mechanism underlying EGFR localization in mitochondria is largely unknown. Using immunofluorescence and immunoelectron microscopy, we observed that EGFR did localize within mitochondria. Moreover, EGFR mitochondrial translocation can be increased by rapamycin treatment in A431 cells and greatly reduced by the presence of 3-methyladenine (3-MA), an inhibitor of autophagy. The reduction of mitochondrial EGFR via autophagy inhibition is further confirmed by small interference RNA (siRNA), through which the essential protein Beclin 1 was depleted. Knocking down Beclin 1 markedly decreased the mitochondrial translocation of EGFR that was induced by rapamycin. We also noticed that the content of mitochondrial EGFR transfer is decreased when the cells are exposed to the apoptotic inducer etoposide. Additionally, either EGF treatment or EGFR knockdown by siRNA results in a greater decline of cell viability in cells possessing more mitochondrial EGFRs. Taken together, we conclude that EGFR mitochondrial localization is regulated by either autophagy or programmed cell death and is correlated with cell survival.

Modulation of survival pathways in ovarian carcinoma cell lines resistant to platinum compounds

Because cytotoxic stress elicits various signaling pathways that may be implicated in cell survival or cell death, their alterations may have relevance in the development of platinum-resistant phenotype. Thus, in the present study, we investigated cell response to the epidermal growth factor receptor (EGFR) inhibitor gefitinib of ovarian carcinoma cell lines, including cells selected for resistance to cisplatin (IGROV-1/Pt1) and oxaliplatin (IGROV-1/OHP). Resistant sublines exhibited a marked decrease in sensitivity to gefitinib and resistance to apoptosis. Gefitinib was capable of inhibiting the phosphorylation of EGFR in all the studied cell lines. The Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) kinases, which act downstream of EGFR, were constitutively active in the three cell lines, but phospho-ERK1/2 levels were increased in the two resistant sublines. This feature was associated with reduced sensitivity to the MEK1/2 inhibitor U0126. Pretreatment of resistant cells with U0126 resulted in restoration of sensitivity to gefitinib. Gefitinib was more effective in inhibiting ERK1/2 and Akt phosphorylation in IGROV-1 cells than in IGROV-1/OHP and IGROV-1/Pt1 cells. Phospho-p38 was up-regulated in the resistant sublines, indicating the concomitant activation of distinct mitogen-activated protein kinases. The up-regulation of phospho-p38 was associated with a peculiar localization of EGFR, which, in resistant sublines, was mainly internalized. In conclusion, our results indicate that the development of resistance to platinum drugs is associated with multiple alterations including deregulation of survival pathways activated by EGFR resulting in a reduced cellular response to gefitinib.

Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis

In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly, the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators of spermatogenesis in bovine.

Characterization of a Novel Tripartite Nuclear Localization Sequence in the EGFR Family

Aberrant expression of epidermal growth factor receptor (EGFR) is present in many human tumors. Several reports have shown that EGFR is translocated into the nucleus during liver regeneration and in several types of cells and tissues such as placenta and thyroid. Nuclear EGFR is associated with transcription, DNA synthesis, and DNA repair activity and serves as a prognostic marker in breast carcinoma and oropharyngeal squamous cell cancer. However, the nuclear localization sequence (NLS) of EGFR has not been extensively examined. In this study, we have shown that the juxtamembrane region of EGFR harbors a putative NLS with three clusters of basic amino acids (RRRHIVRKRTLRR (amino acids 645-657)) that mediates the nuclear localization of EGFR. We found that this newly characterized tripartite NLS is conserved among the EGFR family members (EGFR, ErbB2, ErbB3, and ErbB4) and is able to move each to the nucleus. Further, this tripartite NLS could also mediate the nuclear localization of other known cytoplasmic proteins such as pyruvate kinase. We have demonstrated that mutating one of the three basic amino acid clusters (R or K --> A) leads to significant impairment of the nuclear localization of EGFR and that of a green fluorescent protein-pyruvate kinase-NLS reporter protein. Our results show that this tripartite NLS is distinct from the traditional mono- and bipartite NLS and reveal a mechanism that could account for the nuclear localization of membrane receptors.

Heparin‐binding epidermal growth factor‐like growth factor isoforms and epidermal growth factor receptor/ErbB1 expression in bladder cancer and their relation to clinical outcome

Cleavage of membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields a soluble HB-EGF isoform (sHB-EGF), which is an activating epidermal growth factor receptor (EGFR) ligand and a C-terminal fragment HB-EGF-C acting directly in the nucleus. In bladder cancer, overexpression of both HB-EGF and EGFR have been observed, but to the authors' knowledge the prognostic significance of different modes of HB-EGF signaling have remained unclear. Expression and intracellular localization of HB-EGF and EGFR were examined by immunohistochemistry in paraffin-embedded specimens from 121 patients who underwent cystectomy for bladder cancer. Tumor stage was pTis/pT1 in 7 patients, pT2 in 41 patients, pT3 in 55 patients, and pT4 in 18 patients. Lymph node metastases were present in 32 patients. Using an antibody directed against the C-terminal domain, HB-EGF expression was detected in the cytoplasm or in the nucleus of tumor cells. EGFR staining was uniform at the plasma membrane. The actuarial 5-year cancer-specific survival of patients with tumors with predominant nuclear HB-EGF staining was 28% compared with 57% if HB-EGF staining was predominantly cytoplasmic (P = .027). Disease outcome of patients with a 'mixed' HB-EGF staining pattern was found to be between that of the 2 former groups. In agreement with previous studies, strong EGFR expression was associated with poor prognosis. Despite strong EGFR expression, predominant cytoplasmic HB-EGF staining was associated with a more favorable outcome, whereas a predominant nuclear pattern defined a subgroup with extremely poor prognosis (5-year tumor-specific survival of 55% vs 13%, respectively; P = .026). The current study results confirm that EGFR expression is significantly correlated with disease-specific mortality but that the outcome is also influenced by the mode of HB-EGF signaling. Additional nuclear HB-EGF signaling, indicative of increased cleavage of proHB-EGF, appears to enhance the adverse activities. Cytoplasmic HB-EGF staining likely reflects proHB-EGF, which may also exert antiproliferative effects.

Role of the Sec61 Translocon in EGF Receptor Trafficking to the Nucleus and Gene Expression

The epidermal growth factor (EGF)-dependent trafficking of the intact EGF receptor to the nucleus and its requirement for growth factor induction of cyclin D and other genes has been reported. Unresolved is the mechanism by which this or other transmembrane proteins are excised from a lipid bilayer before nuclear translocalization. We report that, after the addition of EGF, the cell surface EGF receptor is trafficked to the endoplasmic reticulum (ER) where it associates with Sec61beta, a component of the Sec61 translocon, and is retrotranslocated from the ER to the cytoplasm. Abrogation of Sec61beta expression prevents EGF-dependent localization of EGF receptors to the nucleus and expression of cyclin D. This indicates that EGF receptors are trafficked from the ER to the nucleus by a novel pathway that involves the Sec61 translocon.

Oridonin Inhibited the Tyrosine Kinase Activity and Induced Apoptosis in Human Epidermoid Carcinoma A431 Cells

Oridonin, an active component isolated from the plant Rabdosia rubescens, has been reported to exhibit antitumor effects, but little is known about its molecular mechanism of action. In this study, we first investigated the mechanism involved in oridonin-induced cell death in human epidermoid carcinoma A431 cells, which overexpress epidermal growth factor receptor (EGFR). After treatment with various doses of oridonin for 24 h, the majority of A431 cells underwent apoptosis in a time- and dose-dependent manner as measured by an LDH activity-based assay. Treatment with oridonin at various concentrations for 24 h caused significant inhibition on the total tyrosine kinase activities and downregulation of EGFR expression or EGFR phosphorylation. Oridonin significantly affected the localization of EGFR and phosphorylated EGFR on the cell membrane. However, genistein (a well-known tyrosine kinase inhibitor) did not induce apoptotic A431 cell death. Importantly, oridonin exhibited much stronger inhibitory effect on the total tyrosine kinase activities or EGFR tyrosine phosphorylation as well as much stronger suppression on EGFR and phosphorylated EGFR localization than genistein in A431 cells. Taken together, oridonin exerted a potential inhibitory effect on the tyrosine kinase activity of A431 cells. The decrease in the tyrosine kinase activity and the blockage of EGFR tyrosine phosphorylation might be one of the causes of oridonin-induced A431 cell death.

Cell fate-specific regulation of EGF receptor trafficking during Caenorhabditis elegans vulval development

By controlling the subcellular localization of growth factor receptors, cells can modulate the activity of intracellular signal transduction pathways. During Caenorhabditis elegans vulval development, a ternary complex consisting of the LIN-7, LIN-2 and LIN-10 PDZ domain proteins localizes the epidermal growth factor receptor (EGFR) to the basolateral compartment of the vulval precursor cells (VPCs) to allow efficient receptor activation by the inductive EGF signal from the anchor cell. We have identified EGFR substrate protein-8 (EPS-8) as a novel component of the EGFR localization complex that links receptor trafficking to cell fate specification. EPS-8 expression is upregulated in the primary VPCs, where it creates a positive feedback loop in the EGFR/RAS/MAPK pathway. The membrane-associated guanylate kinase LIN-2 recruits EPS-8 into the receptor localization complex to retain the EGFR on the basolateral plasma membrane, and thus allow maximal receptor activation in the primary cell lineage. Low levels of EPS-8 in the neighboring secondary VPCs result in the rapid degradation of the EGFR, allowing these cells to adopt the secondary cell fate. Extracellular signals thus regulate EGFR trafficking in a cell type-specific manner to control pattern formation during organogenesis.

Potential crosstalk between insulin-like growth factor receptor type 1 and epidermal growth factor receptor in progression and metastasis of pancreatic cancer

The insulin-like growth factor receptor type 1 (IGF1R) and epidermal growth factor receptor (EGFR) are reportedly overexpressed in pancreatic cancer. However, the correlation between activated EGFR and IGF1R and their clinicopathological implications still remain unclear. The cellular localization and overexpression of IGF1R and EGFR were investigated immunohistochemically in primary invasive ductal pancreatic carcinomas obtained from 74 patients who underwent radical surgical resection. We also compared the status of IGF1R and EGFR overexpression between primary tumors and hepatic metastatic tumors obtained from 44 autopsied patients. Among the 74 surgically resected primary tumors, cytoplasm- and membrane-dominant EGFR overexpression was detected in 22 (30%) and 7 (9%), respectively, whereas cytoplasm- and membrane-dominant IGF1R overexpression was detected in 8 (11%) and 28 (38%), respectively. Membrane-dominant EGFR and cytoplasm-dominant IGF1R were more frequent in lower-grade tumors and correlated with favorable prognosis, whereas cytoplasm-dominant EGFR and membrane-dominant IGF1R were more frequent in higher-grade tumors and correlated with poor prognosis. In 36 autopsy specimens of pancreatic tumor with concurrent overexpression of IGF1R and EGFR, there was an inverse correlation between the IGF1R and EGFR localization patterns (P=0.001). In the hepatic metastatic tumors obtained by autopsy, the incidences of both IGF1R and EGFR overexpression were much higher than in the surgically resected primary tumors. More than half of the autopsy cases consistently showed membrane-dominant EGFR expression in both the primary tumor and hepatic metastases, whereas IGF1R expression showed considerable variation. Crosstalk between differently localized IGF1R and EGFR might play a role in determining the biological aggressiveness of pancreatic cancer, although their cellular localization may often alter during the process of metastasis.

Rab7 Activity Affects Epidermal Growth Factor:Epidermal Growth Factor Receptor Degradation by Regulating Endocytic Trafficking from the Late Endosome

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family. Ligand (epidermal growth factor or EGF) binding to the EGFR results in the coordinated activation and integration of biochemical signaling events to mediate cell growth, migration, and differentiation. One mechanism the cell utilizes to orchestrate these events is ligand-mediated endocytosis through the canonical clathrin-mediated endocytic pathway. Identification of proteins that regulate the intracellular movement of the EGF.EGFR complex is an important first step in dissecting how specificity of EGFR signaling is conferred. We examined the role of the small molecular weight guanine nucleotide-binding protein (G-protein) rab7 as a regulator of the distal stages of the endocytic pathway. Through the transient expression of activating and inactivating mutants of rab7 in HeLa cells, we have determined that rab7 activity directly correlates with the rate of radiolabeled EGF and EGFR degradation. Furthermore, when inhibitory mutants of rab7 are expressed, the internalized EGF.EGFR complex accumulates in high-density endosomes that are characteristic of the late endocytic pathway. Thus, we conclude that rab7 regulates the endocytic trafficking of the EGF.EGFR complex by regulating its lysosomal degradation.

Epidermal growth factor potentiates renal cell death in hydronephrotic neonatal mice, but cell survival in rats

Epidermal growth factor (EGF) markedly attenuates tubular apoptosis induced by unilateral ureteral obstruction (UUO) in the neonatal rat, and reduces apoptosis induced by mechanical stretch of cultured rat tubular cells. To investigate the role of EGF in modulating apoptosis resulting from UUO, neonatal wild type and mutant mice lacking EGF (knockout), or with diminished EGF receptor activity (waved-2 mutant) were compared to control mice for tubular apoptosis and atrophy. Rat and mouse kidneys were compared for localization of the EGF receptor. Apoptosis was also measured in cultured mouse tubular cells subjected to stretch and exposed to EGF. UUO reduced endogenous renal EGF expression in wild-type mice. Unlike the rat, exogenous EGF did not decrease tubular apoptosis or atrophy in the obstructed kidney, and significantly increased stretch-induced apoptosis of cultured mouse tubular cells. Tubular apoptosis was 50% lower in the obstructed kidney of EGF knockout and waved-2 mice relative to wild type and heterozygous animals. Exogenous EGF increased tubular apoptosis and doubled atrophy in the obstructed kidney of waved-2 mice. Species differences in EGF receptor localization were detected in 3-day-old kidneys. EGF acts as a survival factor in the neonatal rat, but potentiates tubular cell death in the neonatal mouse. Species differences are maintained in cultured cells, suggesting that differences in EGF receptor signaling underlie these opposing effects.

Over-expression of GM1 enhances cell proliferation with epidermal growth factor without affecting the receptor localization in the microdomain in PC12 cells

Sialic acid-containing glycosphingolipids, gangliosides, are expressed at high levels in the nerve tissues and various tumor cells. Although a number of studies on the roles of gangliosides in the regulation of cell proliferation have been performed, the mechanisms for the regulation are not well understood. We established PC12 transfectant cells over-expressing GM1 using cloned beta1,3-galactosyltransferase (EC: 2.4.1.62) cDNA, and analyzed their growth and growth signals with epidermal growth factor (EGF). Over-expression of GM1 enhanced the cell proliferation with EGF under low serum culture conditions. The phosphorylation levels of EGF receptor and downstream MAP kinases after EGF stimulation were sustained even after 60 min in the transfectant cells. In contrast with Swiss3T3 cells, in which we previously reported growth suppression with GM1 over-expression due to a dramatic change in the intracellular localization of PDGF receptor, PC12 transfectant cells with beta1,3-galactosyltransferase cDNA showed no clear changes in the intracellular localization of EGF receptor in the microdomain/raft fractionation experiments compared with the vector control cells. These results suggested that the effects of GM1 expression on the nature of microdomains and growth signals depend on the cell types and receptors analyzed.

Continual expression of Rab5(Q79L) causes a ligand-independent EGFR internalization and diminishes EGFR activity.

The amount of cell-surface Epidermal Growth Factor Receptor (EGFR) available to secreted ligand (EGF) dictates a cell's ability to mediate cell proliferation, differentiation or migration. Multiple factors regulate EGFR cell-surface expression including the rates of protein synthesis and protein degradation, and the endocytic trafficking of both stimulated and unstimulated EGFR. Rab5 is a 25 kDa protein that is localized to the plasma membrane and the early endosome. Its exact molecular function, however, remains controversial. We have used stable and transient expression systems in HeLa cells to examine the consequence of continual, overexpression of wild-type and activated mutants of rab5 on EGFR localization and signaling. Continual expression of constitutively activated mutants of rab5 causes a ligand-independent redistribution of EGFRs into intracellular vesicles that can not be blocked with an antagonistic antibody. The net result is a decrease in the level of cell-surface EGFRs available for ligand stimulation. Thus, rab5 activation regulates EGFR signaling by facilitating the internalization of the unliganded EGFR.