scholarly journals 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.

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.

Subfractionation of the endocytic pathway: isolation of compartments involved in the processing of internalised epidermal growth factor-receptor complexes

1989 ◽  
Vol 94 (4) ◽  
pp. 685-694
Author(s):  
C.E. Futter ◽  
C.R. Hopkins
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Biological Effects ◽  
Growth Factor Receptor ◽  
Endocytic Pathway ◽  
Receptor Complexes ◽  
Epidermal Growth ◽  
Endocytic Vesicles ◽  
Different Parts

The aim of the present study was to isolate different parts of the endocytic pathway in order to examine the role of epidermal growth factor (EGF)-receptor internalisation in mediating the biological effects of EGF. We have used an antibody to the transferrin receptor complexed with colloidal gold to modify the density of the endocytic compartments so that they can be purified by sucrose density centrifugation. Using this technique, we have been able to isolate a highly purified preparation of endocytic vesicles from H.Ep.2 cells that contain internalised EGF. By employing pulse—chase protocols, it is possible to isolate the different parts of the endocytic pathway and show that they are temporally distinct with regard to the processing of EGF. It should now be possible to examine interactions between the EGF receptor and intracellular substrates in different parts of the endocytic pathway.

scholarly journals Non-catalytic activation of phospholipase C-γ 1 in vitro by epidermal growth factor receptor

1993 ◽  
Vol 293 (2) ◽  
pp. 507-511 ◽  
Author(s):  
S M T Hernández-Sotomayor ◽  
G Carpenter
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Catalytic Activation ◽  
Epidermal Growth

To investigate the possible functional role of epidermal growth factor (EGF) receptor-phospholipase C-gamma 1 (PLC-gamma 1) complexes, we have measured PLC-gamma 1 activity in vitro in the absence or presence of purified EGF receptor. Immunoprecipitates of PLC-gamma 1 from control A-431 cells were incubated without or with purified EGF receptor in the absence or presence of ATP. Under these conditions the EGF receptor increased non-tyrosine-phosphorylated PLC-gamma 1 activity 3-4-fold in the absence or presence of ATP, but increased tyrosine-phosphorylated and activated PLC-gamma 1 by only 20-50%. Both basal and autophosphorylated forms of the purified EGF receptor increased the activity of the non-tyrosine-phosphorylated PLC-gamma 1, and stoichiometric levels of purified receptor were required to increase PLC activity. Other tyrosine kinases such as the platelet-derived growth factor receptor and erbB-2, but not the insulin receptor, also stimulated PLC-gamma 1 activity. PLC-gamma 1 activity could be activated with the kinase-negative EGF receptor, but a C-terminal truncated receptor was much less effective. Purified EGF receptor could also activate PLC-beta 1, but with a much decreased potency compared with PLC-gamma 1. Our results suggest that in vitro the EGF receptor can increase PLC-gamma 1 activity independently of tyrosine phosphorylation.

scholarly journals Regulation of Epidermal Growth Factor Receptor Down-Regulation by UBPY-mediated Deubiquitination at Endosomes

2005 ◽  
Vol 16 (11) ◽  
pp. 5163-5174 ◽  
Author(s):  
Emi Mizuno ◽  
Takanobu Iura ◽  
Akiko Mukai ◽  
Tamotsu Yoshimori ◽  
Naomi Kitamura ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Down Regulation ◽  
Epidermal Growth

Ligand-activated receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This “receptor down-regulation” process is crucial to terminate the cell proliferation signals produced by activated receptors. During the process, ubiquitination of the receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-regulation of epidermal growth factor (EGF) receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-regulation by deubiquitinating EGFR on endosomes.

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.

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.

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 Current Understanding of the Emerging Role of Prolidase in Cellular Metabolism

2020 ◽  
Vol 21 (16) ◽  
pp. 5906 ◽  
Author(s):  
Magdalena Misiura ◽  
Wojciech Miltyk
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Intracellular Signaling ◽  
Transforming Growth Factor ◽  
Biological Effects ◽  
Growth Factor Receptor ◽  
Cellular Metabolism ◽  
Interferon Α ◽  
Epidermal Growth

Prolidase [EC 3.4.13.9], known as PEPD, cleaves di- and tripeptides containing carboxyl-terminal proline or hydroxyproline. For decades, prolidase has been thoroughly investigated, and several mechanisms regulating its activity are known, including the activation of the β1-integrin receptor, insulin-like growth factor 1 receptor (IGF-1) receptor, and transforming growth factor (TGF)-β1 receptor. This process may result in increased availability of proline in the mitochondrial proline cycle, thus making proline serve as a substrate for the resynthesis of collagen, an intracellular signaling molecule. However, as a ligand, PEPD can bind directly to the epidermal growth factor receptor (EGFR, epidermal growth factor receptor 2 (HER2)) and regulate cellular metabolism. Recent reports have indicated that PEPD protects p53 from uncontrolled p53 subcellular activation and its translocation between cellular compartments. PEPD also participates in the maturation of the interferon α/β receptor by regulating its expression. In addition to the biological effects, prolidase demonstrates clinical significance reflected in the disease known as prolidase deficiency. It is also known that prolidase activity is affected in collagen metabolism disorders, metabolic, and oncological conditions. In this article, we review the latest knowledge about prolidase and highlight its biological function, and thus provide an in-depth understanding of prolidase as a dipeptidase and protein regulating the function of key biomolecules in cellular metabolism.

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