scholarly journals Phosphorylation of Tyrosine 992, 1068, and 1086 Is Required for Conformational Change of the Human Epidermal Growth Factor Receptor C-Terminal Tail

1999 ◽  
Vol 10 (3) ◽  
pp. 525-536 ◽  
Author(s):  
Anupam Bishayee ◽  
Laura Beguinot ◽  
Subal Bishayee
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Point Mutations ◽  
Growth Factor Receptor ◽  
Binding Activity ◽  
Tyrosine Residues ◽  
Amino Acid Peptide ◽  
C Terminus ◽  
Epidermal Growth

We reported previously that a conformation-specific antibody, Ab P2, to a 16-amino acid peptide (Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg) of the cytoplasmic domain of the β-type platelet-derived growth factor receptor also recognizes the epidermal growth factor (EGF) receptor. Although the antibody is not directed to phosphotyrosine, it recognizes in immunoprecipitation the activated and hence phosphorylated form of both receptors. In P2 peptide, there are two tripeptide sequences, Asp-Glu-Glu and Tyr-Gln-Gln, that are also present in the EGF receptor. Our present studies using either EGF receptor C-terminal deletion mutants or point mutations (Tyr→Phe) and our previous studies on antibody inhibition by P2-derived peptides suggest that Gln-Gln in combination with Asp-Glu-Glu forms a high-affinity complex with Ab P2 and that such complex formation is dependent on tyrosine phosphorylation. Of the five phosphate acceptor sites in the EGF receptor, clustered in the extreme C-terminal tail, phosphorylation of three tyrosine residues (992, 1068, and 1086) located between Asp-Glu-Glu and Gln-Gln is necessary for Ab P2 binding. In contrast, the acceptor sites Tyr 1173 and 1148 play no role in the conformation change. Asp-Glu-Glu and Gln-Gln are located 169 amino acids apart, and it is highly likely that the interactions among three negatively charged phosphotyrosine residues in the receptor C terminus may result in the bending of the peptide chain in such a way that these two peptides come close to each other to form an antibody-binding site. Such a possibility is also supported by our finding that receptor dephosphorylation results in complete loss of Ab P2–binding activity. In conclusion, we have identified a domain within the cytoplasmic part of the EGF receptor whose conformation is altered by receptor phosphorylation; furthermore, we have identified the tyrosine residues that positively regulate this conformation.

scholarly journals Epidermal Growth Factor Receptor Distribution during Chemotactic Responses

2000 ◽  
Vol 11 (11) ◽  
pp. 3873-3883 ◽  
Author(s):  
Maryse Bailly ◽  
Jeffrey Wyckoff ◽  
Boumediene Bouzahzah ◽  
Ross Hammerman ◽  
Vonetta Sylvestre ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Fluorescent Protein ◽  
Growth Factor Receptor ◽  
Spatial Gradient ◽  
Spatial Gradients ◽  
Epidermal Growth ◽  
Green Fluorescent

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.

scholarly journals Activation of the Epidermal Growth Factor (EGF) Receptor Induces Formation of EGF Receptor- and Grb2-Containing Clathrin-Coated Pits

2006 ◽  
Vol 26 (2) ◽  
pp. 389-401 ◽  
Author(s):  
Lene E. Johannessen ◽  
Nina Marie Pedersen ◽  
Ketil Winther Pedersen ◽  
Inger Helene Madshus ◽  
Espen Stang
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Mitogen Activated Protein Kinase ◽  
Coated Pits ◽  
Α Subunit ◽  
Sh3 Domains ◽  
Epidermal Growth

ABSTRACT In HeLa cells depleted of adaptor protein 2 complex (AP2) by small interfering RNA (siRNA) to the μ2 or α subunit or by transient overexpression of an AP2 sequestering mutant of Eps15, endocytosis of the transferrin receptor (TfR) was strongly inhibited. However, epidermal growth factor (EGF)-induced endocytosis of the EGF receptor (EGFR) was inhibited only in cells where the α subunit had been knocked down. By immunoelectron microscopy, we found that in AP2-depleted cells, the number of clathrin-coated pits was strongly reduced. When such cells were incubated with EGF, new coated pits were formed. These contained EGF, EGFR, clathrin, and Grb2 but not the TfR. The induced coated pits contained the α subunit, but labeling density was reduced compared to control cells. Induction of clathrin-coated pits required EGFR kinase activity. Overexpression of Grb2 with inactivating point mutations in N- or C-terminal SH3 domains or in both SH3 domains inhibited EGF-induced formation of coated pits efficiently, even though Grb2 SH3 mutations did not block activation of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K). Our data demonstrate that EGFR-induced signaling and Grb2 are essential for formation of clathrin-coated pits accommodating the EGFR, while activation of MAPK and PI3K is not required.

Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras

1994 ◽  
Vol 14 (3) ◽  
pp. 1575-1581
Author(s):  
G J Pronk ◽  
A M de Vries-Smits ◽  
L Buday ◽  
J Downward ◽  
J A Maassen ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Nucleotide Exchange ◽  
Similar Time ◽  
Epidermal Growth

Shc proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have studied the role of Shc in insulin- and EGF-induced activation of p21ras in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21ras. Analysis of Shc immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in Shc tyrosine phosphorylation and (ii) Shc antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of Shc and the presence of Grb2 and mSOS in Shc immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, Shc could be detected as well. Furthermore, Shc immune complexes contained guanine nucleotide exchange activity toward p21ras in vitro. From these results, we conclude that after insulin and EGF treatment, Shc associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21ras. In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with Shc was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor. These results indicate that the association of Grb2-nSOS with Shc may be an important event in insulin-induced, mSOS-mediated activation of p21ras.

An epidermal growth factor receptor/ret chimera generates mitogenic and transforming signals: evidence for a ret-specific signaling pathway

1994 ◽  
Vol 14 (1) ◽  
pp. 663-675
Author(s):  
M Santoro ◽  
W T Wong ◽  
P Aroca ◽  
E Santos ◽  
B Matoskova ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinases ◽  
Mammalian Cells ◽  
Egf Receptor ◽  
Biological Effects ◽  
Ectopic Expression ◽  
Growth Factor Receptor ◽  
Dependent Signal ◽  
Epidermal Growth

A chimeric expression vector which encoded for a molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) and the intracellular domain of the ret kinase (EGFR/ret chimera) was generated. Upon ectopic expression in mammalian cells, the EGFR/ret chimera was correctly synthesized and transported to the cell surface, where it was shown capable of binding EGF and transducing an EGF-dependent signal intracellularly. Thus, the EGFR/ret chimera allows us to study the biological effects and biochemical activities of the ret kinase under controlled conditions of activation. Comparative analysis of the growth-promoting activity of the EGFR/ret chimera expressed in fibroblastic or hematopoietic cells revealed a biological phenotype clearly distinguishable from that of the EGFR, indicating that the two kinases couple with mitogenic pathways which are different to some extent. Analysis of biochemical pathways implicated in the transduction of mitogenic signals also evidenced significant differences between the ret kinase and other receptor tyrosine kinases. Thus, the sum of our results indicates the existence of a ret-specific pathway of mitogenic signaling.

scholarly journals Asbestos-induced phosphorylation of epidermal growth factor receptor is linked to c-fos and apoptosis

1999 ◽  
Vol 277 (4) ◽  
pp. L684-L693 ◽  
Author(s):  
Christine L. Zanella ◽  
Cynthia R. Timblin ◽  
Andrew Cummins ◽  
Michael Jung ◽  
Jonathan Goldberg ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Direct Interaction ◽  
Mrna Levels ◽  
Binding Studies ◽  
Therapeutic Implications ◽  
Asbestos Fibers ◽  
Epidermal Growth

We examined the mechanisms of interaction of crocidolite asbestos fibers with the epidermal growth factor (EGF) receptor (EGFR) and the role of the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway in early-response protooncogene (c- fos/c- jun) expression and apoptosis induced by asbestos in rat pleural mesothelial (RPM) cells. Asbestos fibers, but not the nonfibrous analog riebeckite, abolished binding of EGF to the EGFR. This was not due to a direct interaction of fibers with ligand, inasmuch as binding studies using fibers and EGF in the absence of membranes showed that EGF did not adsorb to the surface of asbestos fibers. Exposure of RPM cells to asbestos caused a greater than twofold increase in steady-state message and protein levels of EGFR ( P < 0.05). The tyrphostin AG-1478, which inhibits the tyrosine kinase activity of the EGFR, but not the tyrphostin A-10, which does not affect EGFR activity, significantly ameliorated asbestos-induced increases in mRNA levels of c- fos but not of c- jun. Pretreatment of RPM cells with AG-1478 significantly reduced apoptosis in cells exposed to asbestos. Our findings suggest that asbestos-induced binding to EGFR initiates signaling pathways responsible for increased expression of the protooncogene c- fos and the development of apoptosis. The ability to block asbestos-induced elevations in c- fos mRNA levels and apoptosis by small-molecule inhibitors of EGFR phosphorylation may have therapeutic implications in asbestos-related diseases.

scholarly journals Localization of Phospholipase D1 to Caveolin-enriched Membrane via Palmitoylation: Implications for Epidermal Growth Factor Signaling

2002 ◽  
Vol 13 (11) ◽  
pp. 3976-3988 ◽  
Author(s):  
Jung Min Han ◽  
Yong Kim ◽  
Jun Sung Lee ◽  
Chang Sup Lee ◽  
Byoung Dae Lee ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Point Mutations ◽  
Dominant Negative ◽  
Growth Factor Signaling ◽  
Wild Type ◽  
Caveolin 1 ◽  
Epidermal Growth ◽  
Egf Signaling

Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCα mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.

scholarly journals Role of Epidermal Growth Factor Receptor Signaling in RAS-Driven Melanoma

2005 ◽  
Vol 25 (10) ◽  
pp. 4176-4188 ◽  
Author(s):  
Nabeel Bardeesy ◽  
Minjung Kim ◽  
Jin Xu ◽  
Ryung-Suk Kim ◽  
Qiong Shen ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Tumor Regression ◽  
Growth Factor Receptor ◽  
Tumor Model ◽  
Receptor Signaling ◽  
Epidermal Growth ◽  
Egf Family ◽  
Tumor Model System

ABSTRACT The identification of essential genetic elements in pathways governing the maintenance of fully established tumors is critical to the development of effective antioncologic agents. Previous studies revealed an essential role for H-RASV12G in melanoma maintenance in an inducible transgenic model. Here, we sought to define the molecular basis for RAS-dependent tumor maintenance through determination of the H-RASV12G-directed transcriptional program and subsequent functional validation of potential signaling surrogates. The extinction of H-RASV12G expression in established tumors was associated with alterations in the expression of proliferative, antiapoptotic, and angiogenic genes, a profile consistent with the observed phenotype of tumor cell proliferative arrest and death and endothelial cell apoptosis during tumor regression. In particular, these melanomas displayed a prominent RAS-dependent regulation of the epidermal growth factor (EGF) family, leading to establishment of an EGF receptor signaling loop. Genetic complementation and interference studies demonstrated that this signaling loop is essential to H-RASV12G-directed tumorigenesis. Thus, this inducible tumor model system permits the identification and validation of alternative points of therapeutic intervention without neutralization of the primary genetic lesion.

scholarly journals Alix/AIP1 Antagonizes Epidermal Growth Factor Receptor Downregulation by the Cbl-SETA/CIN85 Complex

2004 ◽  
Vol 24 (20) ◽  
pp. 8981-8993 ◽  
Author(s):  
Mirko H. H. Schmidt ◽  
Daniela Hoeller ◽  
Jiuhong Yu ◽  
Frank B. Furnari ◽  
Webster K. Cavenee ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Receptor Internalization ◽  
Interacting Protein ◽  
Low Intensity ◽  
C Terminus ◽  
Epidermal Growth ◽  
Interfering Rna

ABSTRACT The assembly of the Cbl-SETA/CIN85-endophilin complex at the C terminus of the epidermal growth factor receptor (EGFR) following ligand activation mediates its internalization and ubiquitination. We found that the SETA/CIN85-interacting protein Alix/AIP1, which also binds endophilins, modulates this complex. Alix was found to associate indirectly with EGFR, regardless of its activation state, and with ΔEGFR, which signals at low intensity and does not bind Cbls or SETA/CIN85. In agreement with this, Alix interaction did not occur via SETA/CIN85. However, SETA/CIN85 and Alix were capable of mutually promoting their interaction with the EGFR. Increasing the level of Alix weakened the interaction between SETA/CIN85 and Cbl and reduced the tyrosine phosphorylation of c-Cbl and the level of ubiquitination of EGFR, SETA/CIN85, and Cbls. This antagonism of the Cbl-SETA/CIN85 complex by Alix was reflected in its diminution of EGFR internalization. In agreement with this, small interfering RNA-mediated knockdown of Alix promoted EGFR internalization and downregulation. It has been suggested that SETA/CIN85 promotes receptor internalization by recruiting endophilins. However, Alix was also capable of increasing the level of endophilin associated with EGFR, implying that this is not sufficient to promote receptor internalization. We propose that Alix inhibits EGFR internalization by attenuating the interaction between Cbl and SETA/CIN85 and by inhibiting Cbl-mediated ubiquitination of the EGFR.

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