High-affinity Binding of Epidermal Growth Factor (EGF) to EGF Receptor Is Disrupted by Overexpression of Mutant Dynamin (K44A)

Ringerike, Tove; Stang, Espen; Johannessen, Lene E.; Sandnes, Dagny; Levy, Finn Olav; Madshus, Inger Helene

doi:10.1074/jbc.273.27.16639

Cited by 43 publications

(44 citation statements)

References 30 publications

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“…These experiments support the conclusion that the display of EGFR on the cell surface and the ligand binding characteristics of EGFRs are not affected by dynamin loss. However, these conclusions contradict an earlier study demonstrating that overexpression of a dominant interfering dynamin mutant (K44A) prevent high affinity EGF binding and reduces EGF stimulation of EGFR autophosphorylation (20).…”

Section: Resultscontrasting

confidence: 54%

“…Our experiments demonstrate that the expression and display of high and low affinity EGFRs on the cell surface are not affected by dynamin loss. These experiments contradict earlier studies demonstrating that overexpression of a dominant interfering dynamin mutant prevent high affinity EGF binding and reduces EGFR autophosphorylation (20). Ligand induced endocytosis of EGFR is strongly impaired in dynamin depleted fibroblasts stimulated with either low EGF concentrations [1-1.5 ng∕mL, conditions under which internalization of EGFR is primarily driven by clathrin mediated endocytosis (4,8)], or high EGF concentrations (100 ng∕mL, a condition under which EGFR endocytosis is thought to be mediated by both clathrin-dependent and clathrinindependent mechanisms).…”

Section: Discussioncontrasting

confidence: 56%

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Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane

Sousa¹,

Lax²,

Shen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

142

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The role of endocytosis in the control of EGF receptor (EGFR) activation and cell signaling was explored by using mouse fibroblasts in which dynamin was conditionally depleted. Dynamin is a GTPase shown to play an important role in the control clathrin mediated endocytosis of EGFR and other cell surface receptors. In this report, we demonstrate that EGF binding activity and the display of high and low affinity EGFRs on the cell surface are not affected by dynamin depletion. By contrast, dynamin depletion leads to a strong inhibition of EGFR endocytosis, robust enhancement of EGFR autophosphorylation and ubiquitination, and slower kinetics of EGFR degradation. Surprisingly, MAPK stimulation induced by either low or high EGF concentrations is not affected by dynamin depletion. While a similar initial Akt response is detected in control or dynamin depleted fibroblasts, a somewhat more sustained Akt stimulation is detected in the dynamin depleted cells. These experiments demonstrate that dynamin-mediated endocytosis leads to attenuation of EGFR activation and degradation and that stimulation of the MAPK response and Akt activation are primarily mediated by activated EGFR located in the plasma membrane.membrane receptors | tyrosine kinases

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Section: Resultscontrasting

confidence: 54%

Section: Discussioncontrasting

confidence: 56%

Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane

Sousa¹,

Lax²,

Shen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

142

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“…We cannot therefore completely discount a supplementary role for class 1 PI 3-kinase activity in Hrs phosphorylation although the effects of mutations in the FYVE domain argue strongly for a role for hVps34. Expression of dominant-negative dynamin has been shown to change the binding affinity of EGF receptors at the plasma membrane (24). With regard to this latter point, we have used saturating conditions of ligand and while EGF receptor phosphorylation is itself somewhat reduced (Fig.…”

Section: Discussionmentioning

confidence: 99%

Endosomal Localization and Receptor Dynamics Determine Tyrosine Phosphorylation of Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate

Urbé

Mills

Stenmark

et al. 2000

Molecular and Cellular Biology

118

120

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Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a prominent target for tyrosine phosphorylation following the activation of tyrosine kinase receptors (11). It was initially shown to lie downstream of the HGF (scatter factor) receptor c-met, but activation of other tyrosine kinase receptors and by cytokines such as interleukin-2 and granulocyte-macrophage colony-stimulating factor also results in phosphorylation of Hrs (1). It is localized to transferrin receptor-containing endosomes (12) and bears significant similarity (including a FYVE-finger motif and a VHS domain) to the Saccharomyces cerevisiae protein Vps27. Vps27 belongs to the class E set of Vps mutants which are defective in transport from the sorting endosome to the vacuole (2, 22). A highly related protein, Hrs-2, has been shown to interact with SNAP-25 and SNAP-23, homologous proteins which are involved in regulated exocytosis and other intracellular fusion events, respectively (3).Two proteins that bind to Hrs have been identified which have been named STAM (for signal-transducing adapter molecule) and Hrs binding protein (Hbp) (1, 31). They show 53% sequence identity, each bears an SH3 domain, and both are also tyrosine phosphorylated. In T cells, overexpression of Hrs leads to suppression of cytokine-mediated DNA synthesis, while a mutant unable to bind STAM is without effect (1). NIH 3T3 cells stably transfected with mutants of Hbp that lack the SH3 domain or the binding site for Hrs are impaired in degrading internalized platelet-derived growth factor (31). This latter result is consistent with a role for Hrs in regulating transport from early to late endosomes (or multivesicular bodies) proposed by analogy to Vps27 function in yeast.The FYVE domain is a double zinc finger domain that has been shown ...

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“…Therefore, an affinity-modulating protein may be a substrate of the EGF receptor kinase. Recently, high affinity EGF binding was found to be lost in HeLa cells overexpressing a mutant dynamin (K44A) (91), suggesting that this affinitymodulating protein interacts with dynamin.…”

Section: Discussionmentioning

confidence: 99%

Quantification of Short Term Signaling by the Epidermal Growth Factor Receptor

Kholodenko¹,

Demin²,

Moehren³

et al. 1999

Journal of Biological Chemistry

539

606

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During the past decade, our knowledge of molecular mechanisms involved in growth factor signaling has proliferated almost explosively. However, the kinetics and control of information transfer through signaling networks remain poorly understood. This paper combines experimental kinetic analysis and computational modeling of the short term pattern of cellular responses to epidermal growth factor (EGF) in isolated hepatocytes. The experimental data show transient tyrosine phosphorylation of the EGF receptor (EGFR) and transient or sustained response patterns in multiple signaling proteins targeted by EGFR. Transient responses exhibit pronounced maxima, reached within 15-30 s of EGF stimulation and followed by a decline to relatively low (quasi-steady-state) levels. In contrast to earlier suggestions, we demonstrate that the experimentally observed transients can be accounted for without requiring receptor-mediated activation of specific tyrosine phosphatases, following EGF stimulation. The kinetic model predicts how the cellular response is controlled by the relative levels and activity states of signaling proteins and under what conditions activation patterns are transient or sustained. EGFR signaling patterns appear to be robust with respect to variations in many elemental rate constants within the range of experimentally measured values. On the other hand, we specify which changes in the kinetic scheme, rate constants, and total amounts of molecular factors involved are incompatible with the experimentally observed kinetics of signal transfer. Quantitation of signaling network responses to growth factors allows us to assess how cells process information controlling their growth and differentiation.

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High-affinity Binding of Epidermal Growth Factor (EGF) to EGF Receptor Is Disrupted by Overexpression of Mutant Dynamin (K44A)

Cited by 43 publications

References 30 publications

Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane

Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane

Endosomal Localization and Receptor Dynamics Determine Tyrosine Phosphorylation of Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate

Quantification of Short Term Signaling by the Epidermal Growth Factor Receptor

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