Use of the Green Fluorescent Protein as a Quantitative Reporter of Epidermal Growth Factor Receptor-Mediated Gene Delivery

1997 ◽  
Vol 3 (1) ◽  
pp. 53-63 ◽  
Author(s):  
David V. Schaffer ◽  
Rachael L. Neve ◽  
Douglas A. Lauffenburger
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Green Fluorescent Protein ◽  
Growth Factor ◽  
Gene Delivery ◽  
Epidermal Growth Factor ◽  
Fluorescent Protein ◽  
Growth Factor Receptor ◽  
Epidermal Growth ◽  
Green Fluorescent

scholarly journals Intracellular Movement of Green Fluorescent Protein–Tagged Phosphatidylinositol 3-Kinase in Response to Growth Factor Receptor Signaling

1999 ◽  
Vol 146 (4) ◽  
pp. 869-880 ◽  
Author(s):  
Helen Gillham ◽  
Matthew C.H.M. Golding ◽  
Rainer Pepperkok ◽  
William J. Gullick
Keyword(s):  
Green Fluorescent Protein ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Growth Factor Receptor ◽  
Phosphatidylinositol 3 Kinase ◽  
Epidermal Growth ◽  
Green Fluorescent ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase which has been implicated in mitogenesis, protein trafficking, inhibition of apoptosis, and integrin and actin functions. Here we show using a green fluorescent protein–tagged p85 subunit that phosphatidylinositol 3-kinase is distributed throughout the cytoplasm and is localized to focal adhesion complexes in resting NIH-3T3, A431, and MCF-7 cells. Ligand stimulation of an epidermal growth factor receptor/c-erbB-3 chimera expressed in these cells results in a redistribution of p85 to the cell membrane which is independent of the catalytic activity of the enzyme and the integrity of the actin cytoskeleton. The movement is, however, dependent on the phosphorylation status of the erbB-3 chimera. Using rhodamine-labeled epidermal growth factor we show that the phosphatidylinositol 3-kinase and the receptors colocalize in discrete patches on the cell surface. Low concentrations of ligand cause patching only at the periphery of the cells, whereas at high concentrations patches were seen over the whole cell surface. Using green fluorescent protein–tagged fragments of p85 we show that binding to the receptor requires the NH2-terminal part of the protein as well as its SH2 domains.

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 MICAL-like1 mediates epidermal growth factor receptor endocytosis

2011 ◽  
Vol 22 (18) ◽  
pp. 3431-3441 ◽  
Author(s):  
Nancy Abou-Zeid ◽  
Rudy Pandjaitan ◽  
Lucie Sengmanivong ◽  
Violaine David ◽  
Gwenaelle Le Pavec ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Fluorescent Protein ◽  
Coiled Coil ◽  
Growth Factor Receptor ◽  
Time Lapse ◽  
Small Gtpase ◽  
Rab Protein ◽  
Epidermal Growth

Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates epithelial tight junction assembly and polarized membrane transport. Here we report that Molecule Interacting with CasL (MICAL)-like1 (MICAL-L1) interacts with GTP-Rab13 and shares a similar domain organization with MICAL. MICAL-L1 has a calponin homology (CH), LIM, proline rich and coiled-coil domains. It is associated with late endosomes. Time-lapse video microscopy shows that green fluorescent protein–Rab7 and mcherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of a late endosome/lysosome-associated protein but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in the late endosomal compartment. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data suggest that the N-terminal CH domain associates with the C-terminal Rab13 binding domain (RBD) of MICAL-L1. The binding of Rab13 to RBD disrupts the CH/RBD interaction, and may induce a conformational change in MICAL-L1, promoting its activation. Our results provide novel insights into the MICAL-L1/Rab protein complex that can regulate EGFR trafficking at late endocytic pathways.

scholarly journals EGFR forms ligand-independent oligomers that are distinct from the active state

2020 ◽  
Vol 295 (38) ◽  
pp. 13353-13362 ◽  
Author(s):  
Patrick O. Byrne ◽  
Kalina Hristova ◽  
Daniel J. Leahy
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Fluorescent Protein ◽  
Growth Factor Receptor ◽  
Physiological Role ◽  
Active State ◽  
Human Epidermal Growth Factor ◽  
Epidermal Growth ◽  
The Stability

The human epidermal growth factor receptor (EGFR/ERBB1) is a receptor tyrosine kinase (RTK) that forms activated oligomers in response to ligand. Much evidence indicates that EGFR/ERBB1 also forms oligomers in the absence of ligand, but the structure and physiological role of these ligand-independent oligomers remain unclear. To examine these features, we use fluorescence microscopy to measure the oligomer stability and FRET efficiency for homo- and hetero-oligomers of fluorescent protein-labeled forms of EGFR and its paralog, human epidermal growth factor receptor 2 (HER2/ERBB2) in vesicles derived from mammalian cell membranes. We observe that both receptors form ligand-independent oligomers at physiological plasma membrane concentrations. Mutations introduced in the kinase region at the active state asymmetric kinase dimer interface do not affect the stability of ligand-independent EGFR oligomers. These results indicate that ligand-independent EGFR oligomers form using interactions that are distinct from the EGFR active state.

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.

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