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

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.

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

10.32920/ryerson.14644383.v1 ◽

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 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.

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

10.32920/ryerson.14644383 ◽

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 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.

Activation of the Epidermal Growth Factor (EGF) Receptor Induces Formation of EGF Receptor- and Grb2-Containing Clathrin-Coated Pits

10.1128/mcb.26.2.389-401.2006 ◽

2006 ◽

Vol 26 (2) ◽

pp. 389-401 ◽

Cited By ~ 81

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 ◽

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.

An epidermal growth factor receptor/ret chimera generates mitogenic and transforming signals: evidence for a ret-specific signaling pathway

10.1128/mcb.14.1.663-675.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 663-675

Author(s):

M Santoro ◽

W T Wong ◽

P Aroca ◽

E Santos ◽

B Matoskova ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Kinases ◽

Mammalian Cells ◽

Egf Receptor ◽

Biological Effects ◽

Ectopic Expression ◽

Growth Factor Receptor ◽

Dependent Signal ◽

A chimeric expression vector which encoded for a molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) and the intracellular domain of the ret kinase (EGFR/ret chimera) was generated. Upon ectopic expression in mammalian cells, the EGFR/ret chimera was correctly synthesized and transported to the cell surface, where it was shown capable of binding EGF and transducing an EGF-dependent signal intracellularly. Thus, the EGFR/ret chimera allows us to study the biological effects and biochemical activities of the ret kinase under controlled conditions of activation. Comparative analysis of the growth-promoting activity of the EGFR/ret chimera expressed in fibroblastic or hematopoietic cells revealed a biological phenotype clearly distinguishable from that of the EGFR, indicating that the two kinases couple with mitogenic pathways which are different to some extent. Analysis of biochemical pathways implicated in the transduction of mitogenic signals also evidenced significant differences between the ret kinase and other receptor tyrosine kinases. Thus, the sum of our results indicates the existence of a ret-specific pathway of mitogenic signaling.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e05-05-0456 ◽

2005 ◽

Vol 16 (12) ◽

pp. 5832-5842 ◽

Cited By ~ 67

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 ◽

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.

Role of Phosphoinositide 3-Kinase in Activation of Ras and Mitogen-Activated Protein Kinase by Epidermal Growth Factor

10.1128/mcb.19.6.4279 ◽

1999 ◽

Vol 19 (6) ◽

pp. 4279-4288 ◽

Cited By ~ 206

Author(s):

Stefan Wennström ◽

Julian Downward

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Map Kinase ◽

Tyrosine Kinases ◽

Kinase Inhibitors ◽

Egf Receptor ◽

Mitogen Activated Protein Kinase ◽

Ras Activation ◽

Mitogen Activated Protein ◽

ABSTRACT The paradigm for activation of Ras and extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase by extracellular stimuli via tyrosine kinases, Shc, Grb2, and Sos does not encompass an obvious role for phosphoinositide (PI) 3-kinase, and yet inhibitors of this lipid kinase family have been shown to block the ERK/MAP kinase signalling pathway under certain circumstances. Here we show that in COS cells activation of both endogenous ERK2 and Ras by low, but not high, concentrations of epidermal growth factor (EGF) is suppressed by PI 3-kinase inhibitors; since Ras activation is less susceptible than ERK2 activation, PI 3-kinase-sensitive events may occur both upstream of Ras and between Ras and ERK2. However, strong elevation of PI 3-kinase lipid product levels by expression of membrane-targeted p110α is by itself never sufficient to activate Ras or ERK2. PI 3-kinase inhibition does not affect EGF-induced receptor autophosphorylation or adapter protein phosphorylation or complex formation. The concentrations of EGF for which PI 3-kinase inhibitors block Ras activation induce formation of Shc-Grb2 complexes but not detectable EGF receptor phosphorylation and do not activate PI 3-kinase. The activation of Ras by low, but mitogenic, concentrations of EGF is therefore dependent on basal, rather than stimulated, PI 3-kinase activity; the inhibitory effects of LY294002 and wortmannin are due to their ability to reduce the activity of PI 3-kinase to below the level in a quiescent cell and reflect a permissive rather than an upstream regulatory role for PI 3-kinase in Ras activation in this system.

Alternative Pathways of Epidermal Growth Factor Receptor Mediated Contractile Force in NR6 Fibroblasts

10.1115/imece1999-0403 ◽

1999 ◽

Author(s):

Fred D. Allen ◽

Clara F. Asnes ◽

Alan Wells ◽

Elliot L. Elson ◽

Douglas A. Lauffenburger

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Mapk Pathway ◽

Contractile Force ◽

Mitogen Activated Protein Kinase ◽

Egfr Signaling ◽

Signaling Mechanism ◽

Force Increase ◽

Abstract We investigated the contractile force response to epidermal growth factor (EGF) stimulation in 3T3-derived NR6 fibroblast cells in order to determine significant pathways of biochemical signaling that mediate the response. We examined the force generating specificity of the EGF receptor (EGFR) signaling mechanism by using mutant NR6 fibroblasts expressing variations of the EGFR construct. The wild-type (WT) cell presented the complete internalizing EGFR signaling construct while the c’973 cell presented an internalization-defective EGFR construct, and the M721 cell presented a kinase-defective EGFR construct making it signaling inert. Additionally we examined the roles of the phospholipasc C-γ (PLCγ) pathway by using the PLC inhibitor U73122 (1 μM) and the mitogen activated protein kinase (MAPK) pathway using the inhibitor PD98059 (10 μM) in the observed contractile force responses. We found that the WT cells showed a rapid but transient force increase within the first hour post-stimulation and the c’973 showed a more gradual increase in force which it sustained for several hours post-stimulation. Blocking the PLCγ activation in the WT cells reduced the peak force increase by 50% while blocking MAPK did not affect the force development in either WT or c’973 cells.

Endosomal Signaling of Epidermal Growth Factor Receptor Stimulates Signal Transduction Pathways Leading to Cell Survival

10.1128/mcb.22.20.7279-7290.2002 ◽

2002 ◽

Vol 22 (20) ◽

pp. 7279-7290 ◽

Cited By ~ 190

Author(s):

Yi Wang ◽

Steven Pennock ◽

Xinmei Chen ◽

Zhixiang Wang

Keyword(s):

Signal Transduction ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Kinase Inhibitor ◽

Nucleation Site ◽

Signal Transduction Pathways ◽

Egfr Signaling ◽

Endosomal Signaling ◽

ABSTRACT In spite of intensified efforts to understand cell signaling from endosomes, there is no direct evidence demonstrating that endosomal signaling is sufficient to activate signal transduction pathways and no evidence to demonstrate that endosomal signaling is able to produce a biological outcome. The lack of breakthrough is due in part to the lack of means to generate endosomal signals without plasma membrane signaling. In this paper, we report the establishment of a system to specifically activate epidermal growth factor (EGF) receptor (EGFR) when it endocytoses into endosomes. We treated cells with EGF in the presence of AG-1478, a specific EGFR tyrosine kinase inhibitor, and monensin, which blocks the recycling of EGFR. This treatment led to the internalization of nonactivated EGF-EGFR complexes into endosomes. The endosome-associated EGFR was then activated by removing AG-1478 and monensin. During this procedure we did not observe any surface EGFR phosphorylation. We also achieved specific activation of endosome-associated EGFR without using monensin. By using this system, we provided original evidence demonstrating that (i) the endosome can serve as a nucleation site for the formation of signaling complexes, (ii) endosomal EGFR signaling is sufficient to activate the major signaling pathways leading to cell proliferation and survival, and (iii) endosomal EGFR signaling is sufficient to suppress apoptosis induced by serum withdrawal.

Receptor tyrosine kinases mediate epithelial Na+ channel inhibition by epidermal growth factor

AJP Renal Physiology ◽

10.1152/ajprenal.00261.2004 ◽

2005 ◽

Vol 288 (1) ◽

pp. F150-F161 ◽

Cited By ~ 32

Author(s):

Qiusheng Tong ◽

James D. Stockand

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Kinases ◽

Egf Receptor ◽

Receptor Tyrosine Kinases ◽

Na Channel ◽

Distal Nephron ◽

Open Probability ◽

Channel Inhibition ◽

Epidermal growth factor (EGF) decreases Na+ reabsorption across distal nephron epithelia. Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ transport in this portion of the nephron. Abnormal ENaC activity and EGF signaling are both associated with polycystic kidney disease localized to the distal nephron. We tested here whether EGF and other ligands for receptor tyrosine kinases (RTK) decrease ENaC activity. EGF markedly and quickly decreased ENaC activity. The RTK inhibitor erbstatin blocked EGF actions on ENaC and when added alone increased channel activity, uncovering basal suppression by endogenous RTK. The protein tyrosine phosphatase inhibitor vanadate, similar to EGF, decreased ENaC activity. Growth factors and vanadate decreased ENaC activity by decreasing open probability. ENaC was not phosphorylated in response to EGF, indicating that intermediary proteins transduce the inhibitory signal from the EGF receptor (EGFR) to ENaC. We find that neither MAPK 1/2 nor c-Src is signaling intermediaries between EGFR and ENaC. Inhibition of ENaC paralleled decreases in plasma membrane phosphatidylinositol 4,5- bisphosphate levels [PtdIns(4,5)P2] and was abolished by clamping PtdIns(4,5)P2. We conclude that EGF and other ligands for RTK decrease ENaC open probability by decreasing membrane PtdIns(4,5)P2 levels.