scholarly journals Characterization of internalization and endosome formation of epidermal growth factor in transfected NIH-3T3 cells by computerized image-intensified three-dimensional fluorescence microscopy.

1989 ◽  
Vol 109 (5) ◽  
pp. 2105-2115 ◽  
Author(s):  
M Benveniste ◽  
J Schlessinger ◽  
Z Kam
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescence Intensity ◽  
Egf Receptor ◽  
Three Dimensional ◽  
Coated Pits ◽  
Total Fluorescence ◽  
Epidermal Growth ◽  
Digitized Pictures ◽  
3T3 Cell Lines ◽  
Nih 3T3

Computerized image-intensified fluorescence microscopy has been used to quantify routing and subcellular concentrations of rhodaminated EGF (Rh-EGF) during its receptor-mediated endocytosis in two transfected NIH-3T3 cell lines expressing 2 X 10(5) and 1.5 X 10(6) receptors per cell, respectively. A series of images were digitized by focusing at different depths through the volume of a single cell. The digitized pictures were corrected for fluorescence photobleaching, and removal of out-of-focus fluorescence contributions by deconvolution using the point spread function of the microscope optics (Agard, D. A., and J. W. Sedat. 1980. Proc. Soc. Photo-Opt. Instr. Eng. 264:110-117) allowed automatic computer analysis of the time dependence of endosomal vesicle size and fluorescence intensity in a live cell and also enabled the study of isolated vesicles. An increase in the amount of fluorescence bound to the cell surface, either by increasing the number of receptors expressed per cell or the concentration of Rh-EGF in the incubation drop, yielded an increase in the total fluorescence of internalized vesicles without an increase in their number and area. The linear relation between fluorescence intensity and area for vesicles at different times indicates that EGF concentration is conserved. This is compatible with fusion of small vesicles to form larger ones. However, as endocytosis proceeds, a twofold increase in the slope of the fluorescence vs. area plots is observed for larger vesicles, suggesting that active sorting causes the EGF to be concentrated. Alternatively, this factor could be produced by cumulative fluorescence contributions from stacked membranes. Since coated pits are internalized independent of their occupancy with EGF receptor, we propose that endocytosis does not involve a mechanism specifically recognizing occupied receptor but is rather triggered by a global intracellular event.

scholarly journals Overexpression of epidermal growth factor receptor in NIH-3T3-transfected cells slows its lateral diffusion and rate of endocytosis.

1988 ◽  
Vol 106 (6) ◽  
pp. 1903-1909 ◽  
Author(s):  
M Benveniste ◽  
E Livneh ◽  
J Schlessinger ◽  
Z Kam
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Lines ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Lateral Diffusion ◽  
Coated Pits ◽  
Epidermal Growth ◽  
Nih 3T3

Interactions between membrane proteins are believed to be important for the induction of transmembrane signaling. Endocytosis is one of the responses which is regulated by both intracellular and extracellular signals. To study such interactions, we have measured the lateral mobility and rate of endocytosis of epidermal growth factor receptor in three transfected NIH-3T3 cell lines (HER84, HER22, and HER82) expressing 2 X 10(4), 2 X 10(5) and 1.5 X 10(6) EGF-receptors per cell, respectively. Using rhodamine-labeled EGF (Rh-EGF) and rhodamine-labeled monoclonal anti-EGF-receptor antibody (Rh-mAb-108), we measured twofold decreases in the lateral diffusion coefficients for each approximately 10-fold increase in EGF-receptor concentration. Since steric effects cannot account for such dependence, we propose that protein mobility within the membrane, which is determined by the rate of motion between immobile barriers, decreases due to aggregate formation. The rate of endocytosis also decreases twofold between the HER84 (2 X 10(4) receptors/cell) and HER22 (2 X 10(5) receptors/cell) cell lines, suggesting that it is diffusion limited. The comparable rates of endocytosis of the HER82 and HER22 cell lines suggest that at high receptor density endocytosis may be limited by the total number of sites for receptors in coated-pits and by their rate of recycling.

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.

scholarly journals The Inhibitory Effect of ErbB2 on Epidermal Growth Factor-induced Formation of Clathrin-coated Pits Correlates with Retention of Epidermal Growth Factor Receptor-ErbB2 Oligomeric Complexes at the Plasma Membrane

2005 ◽  
Vol 16 (12) ◽  
pp. 5832-5842 ◽  
Author(s):  
Camilla Haslekås ◽  
Kamilla Breen ◽  
Ketil W. Pedersen ◽  
Lene E. Johannessen ◽  
Espen Stang ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Inhibitory Effect ◽  
Coated Pits ◽  
Transfected Cells ◽  
Epidermal Growth

By constructing stably transfected cells harboring the same amount of epidermal growth factor (EGF) receptor (EGFR), but with increasing overexpression of ErbB2, we have demonstrated that ErbB2 efficiently inhibits internalization of ligand-bound EGFR. Apparently, ErbB2 inhibits internalization of EGF-bound EGFR by constitutively driving EGFR-ErbB2 hetero/oligomerization. We have demonstrated that ErbB2 does not inhibit phosphorylation or ubiquitination of the EGFR. Our data further indicate that the endocytosis deficiency of ErbB2 and of EGFR-ErbB2 heterodimers/oligomers cannot be explained by anchoring of ErbB2 to PDZ-containing proteins such as Erbin. Instead, we demonstrate that in contrast to EGFR homodimers, which are capable of inducing new clathrin-coated pits in serum-starved cells upon incubation with EGF, clathrin-coated pits are not induced upon activation of EGFR-ErbB2 heterodimers/oligomers.

scholarly journals Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.

1988 ◽  
Vol 8 (5) ◽  
pp. 1970-1978 ◽  
Author(s):  
I Lax ◽  
A Johnson ◽  
R Howk ◽  
J Sap ◽  
F Bellot ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Alpha ◽  
3T3 Cells ◽  
Epidermal Growth ◽  
Factor Alpha ◽  
Differential Binding ◽  
Nih 3T3

The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors.

Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor

1988 ◽  
Vol 8 (6) ◽  
pp. 2302-2308
Author(s):  
E Livneh ◽  
T J Dull ◽  
E Berent ◽  
R Prywes ◽  
A Ullrich ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Egf Receptor ◽  
Wild Type ◽  
3T3 Cells ◽  
Kinase C ◽  
Epidermal Growth ◽  
Nih 3T3

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.

scholarly journals Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor.

1988 ◽  
Vol 8 (6) ◽  
pp. 2302-2308 ◽  
Author(s):  
E Livneh ◽  
T J Dull ◽  
E Berent ◽  
R Prywes ◽  
A Ullrich ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Egf Receptor ◽  
Wild Type ◽  
3T3 Cells ◽  
Kinase C ◽  
Epidermal Growth ◽  
Nih 3T3

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.

Application of Monte Carlo Integration to Three-Dimensional Fluorescence Spectrometry

1995 ◽  
Vol 49 (9) ◽  
pp. 1239-1242 ◽  
Author(s):  
Yuan Yan ◽  
Jin-Gou Xu ◽  
Zhu-Guang Lin ◽  
Yi-Bin Zhao ◽  
Guo-Zhen Chen
Keyword(s):  
Monte Carlo ◽  
Fluorescence Intensity ◽  
Single Point ◽  
Three Dimensional ◽  
Monte Carlo Integration ◽  
Fluorescence Spectrometry ◽  
Total Fluorescence ◽  
Better Than

In this paper, the sensitivity of three-dimensional fluorescence spectrometry (TDFS) is studied by using the total fluorescence intensity, calculated by Monte Carlo integration, instead of conventional single-point intensity. It has been proven that the sensitivity of the total fluorescence intensity method is nearly one hundred times better than that of the single-point intensity method for the determinations of fluorescein and tryptophan. The mechanism of the method has also been studied.

scholarly journals Association with Membrane Protrusions Makes ErbB2 an Internalization-resistant Receptor

2004 ◽  
Vol 15 (4) ◽  
pp. 1557-1567 ◽  
Author(s):  
Anette M. Hommelgaard ◽  
Mads Lerdrup ◽  
Bo van Deurs
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Egf Receptor ◽  
Immunogold Labeling ◽  
Cross Linking ◽  
Coated Pits ◽  
Membrane Protrusions ◽  
Epidermal Growth ◽  
Bulk Membrane

In contrast to the epidermal growth factor (EGF) receptor, ErbB2 is known to remain at the plasma membrane after ligand binding and dimerization. However, why ErbB2 is not efficiently down-regulated has remained elusive. Basically, two possibilities exist: ErbB2 is internalization resistant or it is efficiently recycled after internalization. By a combination of confocal microscopy, immunogold labeling electron microscopy, and biochemical techniques we show that ErbB2 is preferentially associated with membrane protrusions. Moreover, it is efficiently excluded from clathrin-coated pits and is not seen in transferrin receptor-containing endosomes. This pattern is not changed after binding of EGF, heregulin, or herceptin. The exclusion from coated pits is so pronounced that it cannot just be explained by lack of an internalization signal. Although ErbB2 is a raft-associated protein, the localization of ErbB2 to protrusions is not a result of raft binding. Also, an intact actin cytoskeleton is not required for keeping ErbB2 away from coated pits. However, after efficient cross-linking, ErbB2 is removed from protrusions to occur on the bulk membrane, in coated pits, and in endosomes. These data show that ErbB2 is a remarkably internalization-resistant receptor and suggest that the mechanism underlying the firm association of ErbB2 with protrusions also is the reason for this resistance.

scholarly journals Regulation of epidermal growth factor receptor signaling by clathrin-coated membrane microdomains

2021 ◽  
Author(s):  
Camilo Garay
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Akt Signaling ◽  
Membrane Microdomains ◽  
Egfr Signaling ◽  
Coated Pits ◽  
Akt Phosphorylation ◽  
Epidermal Growth

The phosphatidylinositol-3-kinase (PI3K)-Akt signaling axis controls cell survival, proliferation and metabolism, and is activated by receptor tyrosine kinases (RTKs) such as the epidermal growth factor (EGF) receptor (EGFR). In addition to activation of PI3K-Akt signaling, the binding of EGF to its receptor results in rapid recruitment of EGFR to clathrin-coated pits (CCPs) followed by eventual EGFR internalization. Hence, receptor-proximal activation of signaling intermediates occurs while EGFR resides within CCPs; however, whether CCPs are required for EGFR signaling remains poorly understood. Using a combination of pharmacological inhibition and siRNA gene silencing of clathrin, we have examined how clathrin controls EGF-stimulated activation of Akt. We find that perturbation of clathrin, but not of EGFR endocytosis by perturbation of dynamin leads to disruption of EGF-stimulated Akt phosphorylation. This indicates that clathrin acts in a function separate from its role in endocytosis to regulate EGFR signaling at the plasma membrane. The EGF-stimulated phosphorylation of the signaling intermediate Gab1, but not that of EGFR itself, was also abrogated upon disruption of clathrin. We then utilized total internal reflection fluorescence microscopy (TIRF-M) to examine the hierarchy of recruitment of EGFR signaling components to CCPs. Collectively, these findings suggest a role for clathrin as a central regulator of EGFR signaling leading to Gab1 and Akt phosphorylation.

scholarly journals Regulation of epidermal growth factor receptor signaling by clathrin-coated membrane microdomains

2021 ◽  
Author(s):  
Camilo Garay
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Akt Signaling ◽  
Membrane Microdomains ◽  
Egfr Signaling ◽  
Coated Pits ◽  
Akt Phosphorylation ◽  
Epidermal Growth

The phosphatidylinositol-3-kinase (PI3K)-Akt signaling axis controls cell survival, proliferation and metabolism, and is activated by receptor tyrosine kinases (RTKs) such as the epidermal growth factor (EGF) receptor (EGFR). In addition to activation of PI3K-Akt signaling, the binding of EGF to its receptor results in rapid recruitment of EGFR to clathrin-coated pits (CCPs) followed by eventual EGFR internalization. Hence, receptor-proximal activation of signaling intermediates occurs while EGFR resides within CCPs; however, whether CCPs are required for EGFR signaling remains poorly understood. Using a combination of pharmacological inhibition and siRNA gene silencing of clathrin, we have examined how clathrin controls EGF-stimulated activation of Akt. We find that perturbation of clathrin, but not of EGFR endocytosis by perturbation of dynamin leads to disruption of EGF-stimulated Akt phosphorylation. This indicates that clathrin acts in a function separate from its role in endocytosis to regulate EGFR signaling at the plasma membrane. The EGF-stimulated phosphorylation of the signaling intermediate Gab1, but not that of EGFR itself, was also abrogated upon disruption of clathrin. We then utilized total internal reflection fluorescence microscopy (TIRF-M) to examine the hierarchy of recruitment of EGFR signaling components to CCPs. Collectively, these findings suggest a role for clathrin as a central regulator of EGFR signaling leading to Gab1 and Akt phosphorylation.

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