scholarly journals Grb2 Regulates Internalization of EGF Receptors through Clathrin-coated Pits

2003 ◽  
Vol 14 (3) ◽  
pp. 858-870 ◽  
Author(s):  
Xuejun Jiang ◽  
Fangtian Huang ◽  
Andriy Marusyk ◽  
Alexander Sorkin
Keyword(s):  
Growth Factor ◽  
Hela Cells ◽  
Binding Sites ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Growth Factor Receptor ◽  
Receptor Internalization ◽  
Sequence Motifs ◽  
Coated Pits

The molecular mechanisms of clathrin-dependent internalization of epidermal growth factor receptor (EGFR) are not well understood and, in particular, the sequence motifs that mediate EGFR interactions with coated pits have not been mapped. We generated a panel of EGFR mutants and stably expressed these mutants in porcine aortic endothelial (PAE) cells. Interestingly, mutations of tyrosine phosphorylation sites 1068 and 1086 that interact with growth-factor-receptor-binding protein Grb2 completely abolished receptor internalization in PAE cells. Quantitative analysis of colocalization of EGF-rhodamine conjugate and coated pits labeled with yellow-fluorescent-protein–tagged β2 subunit of clathrin adaptor complex AP-2 revealed that EGFR mutants lacking Grb2 binding sites do not efficiently enter coated pits. The depletion of Grb2 from PAE as well as HeLa cells expressing endogenous EGFRs by RNA interference substantially reduced the rate of EGFR internalization through clathrin-dependent pathway, thus providing the direct evidence for the important role of Grb2 in this process. Overexpression of Grb2 mutants, in which the SH3 domains were either deleted or inactivated by point mutations, significantly inhibited EGFR internalization in both PAE and HeLa cells. These findings indicate that Grb2, in addition to its key function in signaling through Ras, has a major regulatory role at the initial steps of EGFR internalization through clathrin-coated pits. Furthermore, the EGFR mutant lacking Grb2 binding sites did not efficiently recruit c-Cbl and was not polyubiquitinated. The data are consistent with the model whereby Grb2 participates in EGFR internalization through the recruitment of Cbl to the receptor, thus allowing proper ubiquitylation of EGFR and/or associated proteins at the plasma membrane.

Intact Ca(2+)-binding sites are required for targeting of annexin 1 to endosomal membranes in living HeLa cells

2000 ◽  
Vol 113 (22) ◽  
pp. 3931-3938 ◽  
Author(s):  
U. Rescher ◽  
N. Zobiack ◽  
V. Gerke
Keyword(s):  
Hela Cells ◽  
Binding Sites ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Membrane Binding ◽  
Growth Factor Receptor ◽  
Intracellular Distribution ◽  
Live Cells ◽  
Membrane Structures ◽  
Annexin 1

Annexin 1 is a Ca(2+)-regulated membrane binding protein and a major substrate of the epidermal growth factor receptor kinase. Because of its properties and intracellular distribution, the protein has been implicated in endocytic trafficking of the receptor, in particular in receptor sorting occurring in multivesicular endosomes. Up to now, however, the localization of annexin 1 to cellular membranes has been limited to subcellular fractionation and immunocytochemical analyses of fixed cells. To establish its localization in live cells, we followed the intracellular fate of annexin 1 molecules fused to the Green Fluorescent Protein (GFP). We show that annexin 1-GFP associates with distinct, transferrin receptor-positive membrane structures in living HeLa cells. A GFP chimera containing the Ca(2+)/phospholipid-binding protein core of annexin 1 also shows a punctate intracellular distribution, although the structures labeled here do not resemble early but, at least in part, late endosomes. In contrast, the cores of annexins 2 and 4 fused to GFP exhibit a cytoplasmic or a different punctate distribution, respectively, indicating that the highly homologous annexin core domains carry distinct membrane specificities within live cells. By inactivating the three high-affinity Ca(2+) binding sites in annexin 1 we also show that endosomal membrane binding of the protein in live HeLa cells depends on the integrity of these Ca(2+) binding sites. More detailed analysis identifies a single Ca(2+) site in the second annexin repeat that is crucially involved in establishing the membrane association. These results reveal for the first time that intracellular membrane binding of an annexin in living cells requires Ca(2+) and is mediated in part through an annexin core domain that is capable of establishing specific interactions.

scholarly journals Ubiquitin ligase SMURF2 enhances epidermal growth factor receptor stability and tyrosine-kinase inhibitor resistance

2020 ◽  
Vol 295 (36) ◽  
pp. 12661-12673 ◽  
Author(s):  
Paramita Ray ◽  
Krishnan Raghunathan ◽  
Aarif Ahsan ◽  
Uday Sankar Allam ◽  
Shirish Shukla ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Molecular Mechanisms ◽  
Growth Factor Receptor ◽  
Receptor Internalization ◽  
Egfr Mutations ◽  
Epidermal Growth

The discovery of activating epidermal growth factor receptor (EGFR) mutations spurred the use of EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib, as the first-line treatment of lung cancers. We previously reported that differential degradation of TKI-sensitive (e.g. L858R) and resistant (T790M) EGFR mutants upon erlotinib treatment correlates with drug sensitivity. We also reported that SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. However, the molecular mechanisms involved remain unclear. Here, using in vitro and in vivo ubiquitination assays, MS, and superresolution microscopy, we show SMURF2–EGFR functional interaction is important for EGFR stability and response to TKI. We demonstrate that L858R/T790M EGFR is preferentially stabilized by SMURF2-UBCH5 (an E3-E2)-mediated polyubiquitination. We identified four lysine residues as the sites of ubiquitination and showed that replacement of one of them with acetylation-mimicking glutamine increases the sensitivity of mutant EGFR to erlotinib-induced degradation. We show that SMURF2 extends membrane retention of EGF-bound EGFR, whereas SMURF2 knockdown increases receptor sorting to lysosomes. In lung cancer cell lines, SMURF2 overexpression increased EGFR levels, improving TKI tolerance, whereas SMURF2 knockdown decreased EGFR steady-state levels and sensitized lung cancer cells. Overall, we propose that SMURF2-mediated polyubiquitination of L858R/T790M EGFR competes with acetylation-mediated receptor internalization that correlates with enhanced receptor stability; therefore, disruption of the E3-E2 complex may be an attractive target to overcome TKI resistance.

Internalization of the epidermal growth factor receptor: role in signalling

2001 ◽  
Vol 29 (4) ◽  
pp. 480-484 ◽  
Author(s):  
A. Sorkin
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
Mitogen Activated Protein Kinase ◽  
Receptor Complexes ◽  
Epidermal Growth

The interaction of the activated epidermal growth factor (EGF) receptor (EGFR) with the Src homology 2 (SH2) domain of Grb2 (growth-factor-receptor-bound protein 2) initiates signalling through Ras and mitogen-activated protein kinase. Grb2 can bind EGFR directly or through another SH2-containing protein, She. Activation of EGFRs by ligand also triggers rapid endocytosis of EGF-receptor complexes. To analyse the spatial and temporal regulation of EGFR interactions with SH2 domains in living cells, we have combined imaging microscopy with a modified method of measuring fluorescence resonance energy transfer (FRET) on a pixel-by-pixel basis using EGFR fused to cyan fluorescent protein (CFP) in pair with Grb2 or She fused to yellow fluorescent protein (YFP). Stimulation by EGF resulted in the recruitment of Grb2-YFP and YFP-Shc to cellular compartments that contained EGFR-CFP, and a large increase in the FRET signal. In particular, FRET measurements indicated that activated EGFR-CFP interacted with YFP-Shc and Grb2-YFP in membrane ruffles and endosomes. These results demonstrate that signalling via EGFRs can occur in the endosomal compartment. Moreover, in contrast with previous biochemical studies, FRET experiments show that a large pool of Grb2 and Shc is associated with EGFRs for a prolonged period after EGF stimulation.

scholarly journals Coordinated Traffic of Grb2 and Ras during Epidermal Growth Factor Receptor Endocytosis Visualized in Living Cells

2002 ◽  
Vol 13 (5) ◽  
pp. 1522-1535 ◽  
Author(s):  
Xuejun Jiang ◽  
Alexander Sorkin
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Specific Interaction ◽  
Growth Factor Receptor ◽  
Receptor Complexes ◽  
Epidermal Growth

Activation of the epidermal growth factor receptor (EGFR) triggers multiple signaling pathways and rapid endocytosis of the epidermal growth factor (EGF)–receptor complexes. To directly visualize the compartmentalization of molecules involved in the major signaling cascade, activation of Ras GTPase, we constructed fusions of Grb2, Shc, H-Ras, and K-Ras with enhanced cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), and used live-cell fluorescence imaging microscopy combined with the fluorescence resonance energy transfer (FRET) technique. Stimulation of cells by EGF resulted in the accumulation of large pools of Grb2-CFP and YFP-Shc in endosomes, where these two adaptor proteins formed a complex with EGFR. H-Ras and K-Ras fusion proteins were found at the plasma membrane, particularly in ruffles and lamellipodia, and also in endosomes independently of GTP/GDP loading and EGF stimulation. The relative amount of endosomal H-Ras was higher than that of K-Ras, whereas K-Ras predominated at the plasma membrane. On application of EGF, Grb2, and Ras converge in the same endosomes through the fusion of endosomes containing either Grb2 or Ras or through the joint internalization of two proteins from the plasma membrane. To examine the localization of the GTP-bound form of Ras, we used a FRET assay that exploits the specific interaction of GTP-bound CFP-Ras with the YFP-fused Ras binding domain of c-Raf. FRET microscopy revealed that GTP-bound Ras is located at the plasma membrane, mainly in ruffles and at the cell edges, as well as in endosomes containing EGFR. These data point to the potential for endosomes to serve as sites of generation for persistent signaling through Ras.

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 Molecular mechanisms of epidermal growth factor receptor overexpression in patients with cervical cancer

2011 ◽  
Vol 24 (5) ◽  
pp. 720-728 ◽  
Author(s):  
Marlies Schrevel ◽  
Arko Gorter ◽  
Sandra M Kolkman-Uljee ◽  
J Baptist M Z Trimbos ◽  
Gert Jan Fleuren ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Molecular Mechanisms ◽  
Growth Factor Receptor ◽  
Epidermal Growth
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