scholarly journals Transit of epidermal growth factor through coated pits of the Golgi system.

1982 ◽  
Vol 94 (1) ◽  
pp. 207-212 ◽  
Author(s):  
M C Willingham ◽  
I H Pastan
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Carcinoma Cells ◽  
Coated Pits ◽  
Human Carcinoma ◽  
Golgi System ◽  
Reticular System ◽  
Entire Cell ◽  
Epidermal Growth

Using the direct conjugate of epidermal growth factor (EGF) and horseradish peroxidase, we have followed the entry of EGF into KB (human carcinoma) cells. EGF initially was found bound diffusely to the entire cell surface at 4 degrees C; on warming to 37 degrees C, EGF was found clustered in clathrin-coated pits on the plasma membrane in 1 min or less. Within 1-2 min at 37 degrees C, EGF began to accumulate in receptosomes within the cell and remained there for up to 10 min. At 10-13 min after warming to 37 degrees C, EGF was found in thin reticular membranous elements of the Golgi system, as well as concentrated in the clathrin-coated pits present on these membranes. By 15 min after warming, EGF began to be delivered to lysosomes located near the Golgi system. These findings suggest that clathrin-coated pits in the Golgi reticular system accumulate EGF before delivery to lysosomes.

scholarly journals Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431.

1979 ◽  
Vol 81 (2) ◽  
pp. 382-395 ◽  
Author(s):  
H T Haigler ◽  
J A McKanna ◽  
S Cohen
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Carcinoma Cells ◽  
Biologically Active ◽  
Molar Ratio ◽  
Multivesicular Bodies ◽  
Egf Receptors ◽  
Human Carcinoma ◽  
Epidermal Growth

We have prepared a conjugate of epidermal growth factor (EGF) and ferritin that retains substantial binding affinity for cell receptors and is biologically active. Glutaraldehyde-activated EGF was covalently linked to ferritin to produce a conjugate that contained EGF and ferritin in a 1:1 molar ratio. The conjugate was separated from free ferritin by affinity chromatography using antibodies to EGF. Monolayers of human epithelioid carcinoma cells (A-431) were incubated with EGF:ferritin at 4 degrees C and processed for transmission electron microscopy. Under these conditions, approximately 6 X 10(5) molecules of EGF:ferritin bound to the plasma membrane of each cell. In the presence of excess native EGF, the number of bound ferritin particles was reduced by 99%, indicating that EGF:ferritin binds specifically to cellular EGF receptors. At 37 degrees C, cell-bound EGF:ferritin rapidly redistributed in the plane of the plasma membrane to form small groups that were subsequently internalized into pinocytic vesicles. By 2.5 min at 37 degrees C, 32% of the cell-bound EGF:ferritin was localized in vesicles. After 2.5 min, there was a decrease in the proportion of conjugate in vesicles with a concomitant accumulation of EGF:ferritin in multivesicular bodies. By 30 min, 84% of the conjugate was located in structures morphologically identified as multivesicular bodies or lysosomes. These results are consistent with other morphological and biochemical studies utilizing 125I-EGF and fluorescein-conjugated EGF.

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 Rapid stimulation of pinocytosis in human carcinoma cells A-431 by epidermal growth factor.

1979 ◽  
Vol 83 (1) ◽  
pp. 82-90 ◽  
Author(s):  
H T Haigler ◽  
J A McKanna ◽  
S Cohen
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Biological Activities ◽  
Fluid Phase ◽  
Fold Increase ◽  
Carcinoma Cells ◽  
Tumor Promoter ◽  
Metabolic Inhibitors ◽  
Human Carcinoma ◽  
Epidermal Growth

Horseradish peroxidase (HRP) uptake was used to measure fluid-phase pinocytosis in monolayers of human epithelioid carcinoma cells (A-431). Histochemistry confirmed that cell-associated HRP was restricted to intracellular vesicles. Biochemical methods showed that HRP uptake in control cultures was directly proportional to the duration of exposure. The addition of low concentrations of epidermal growth factor (EGF) to the incubation media produced a 10-fold increase in the initial rate of pinocytosis. The EGF effect was rapid (within 30 s) but transient; the rate of pinocytosis returned to control levels within 15 min. Metabolic inhibitors reduced the EGF-stimulated rate of pinocytosis by greater than 90%. A conjugate of EGF and ferritin (F:EGF) was used to simultaneously compare the intracellular locations of EGF and HRP. Much of F:EGF was internalized in approximately 100-nm vesicles, while most of the HRP was located in much larger vesicles (range 0.1--1.2 micrometer) which also contained F:EGF. The tumor-promoter 12-0-tetradecanoyl-phorbol-13-acetate, which shares several biological activities with EGF, was also effective in stimulating an increase in the rate of pinocytosis.

scholarly journals Clathrin and TOM1L1 regulate epidermal growth factor receptor signaling at the plasma membrane

2021 ◽  
Author(s):  
Gurjeet Singh Judge
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Intracellular Signaling ◽  
Fluorescent Labeling ◽  
Cell Physiology ◽  
Egfr Signaling ◽  
Coated Pits ◽  
Akt Phosphorylation ◽  
Epidermal Growth

Epidermal growth factor (EGF) receptor (EGFR) controls many aspects of cell physiology via the activation of intracellular signaling pathways. Aberrant EGFR signaling and overexpression of EGFR is a characteristic of various cancer types. Ligandbound EGFR elicits phosphorylation of Gab1, which activates phosphatidylinositol-3-kinase (PI3K), which in turn leads to Akt phosphorylation and activation. Akt is a central regulator of cell survival and metabolism. Rapidly upon binding EGF, the receptor is recruited to clathrin-coated pits (CCPs), eventually leading to EGFR endocytosis. As such, receptor-proximal EGFR signaling occurs while the receptor is enriched with CCPs at the plasma membrane. We have recently found that clathrin, but not receptor endocytosis, is required for EGF-stimulated phosphorylation of Akt, yet how clathrin controls EGFR signaling at the plasma membrane remains poorly understood. Clathrin interacts with and recruits numerous proteins to the plasma membrane. To examine how specific clathrin-interacting proteins may underlie the requirement for clathrin in EGFR signaling, I have examined the role of TOM1L1, which interacts with clathrin, EGFR and Src-family kinases (SFKs) through distinct domains. SiRNA gene silencing of TOM1L1 impairs EGF-stimulated Akt phosphorylation, as did expression of a dominant-interfering mutant of TOM1L1 deficient in clathrin binding. Using fluorescent labeling of clathrin, TOM1L1 and other signaling intermediates coupled to total internal reflection fluorescence microscopy; I found that CCPs are enriched in TOM1L1 and phosphorylated Gab1 upon EGF stimulation. These findings indicate that the clathrin-binding adaptor TOM1L1 regulates EGF-stimulated Akt phosphorylation, thus revealing a novel molecular mechanism of regulation of EGFR signaling at the plasma membrane.

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