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.

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.

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.

scholarly journals Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

2015 ◽  
Vol 26 (19) ◽  
pp. 3504-3519 ◽  
Author(s):  
Camilo Garay ◽  
Gurjeet Judge ◽  
Stefanie Lucarelli ◽  
Stephen Bautista ◽  
Rohan Pandey ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Gene Silencing ◽  
Receptor Internalization ◽  
Membrane Structures ◽  
Coated Pits ◽  
Akt Phosphorylation ◽  
Erbb2 Expression ◽  
Epidermal Growth

Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation.

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 Selective regulation of clathrin-mediated epidermal growth factor receptor signaling and endocytosis by phospholipase C and calcium

2017 ◽  
Vol 28 (21) ◽  
pp. 2802-2818 ◽  
Author(s):  
Ralph Christian Delos Santos ◽  
Stephen Bautista ◽  
Stefanie Lucarelli ◽  
Leslie N. Bone ◽  
Roya M. Dayam ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Surface Protein ◽  
Egfr Signaling ◽  
Coated Pits ◽  
Cellular Processes ◽  
Phospholipase Cγ1 ◽  
Epidermal Growth

Clathrin-mediated endocytosis is a major regulator of cell-surface protein internalization. Clathrin and other proteins assemble into small invaginating structures at the plasma membrane termed clathrin-coated pits (CCPs) that mediate vesicle formation. In addition, epidermal growth factor receptor (EGFR) signaling is regulated by its accumulation within CCPs. Given the diversity of proteins regulated by clathrin-mediated endocytosis, how this process may distinctly regulate specific receptors is a key question. We examined the selective regulation of clathrin-dependent EGFR signaling and endocytosis. We find that perturbations of phospholipase Cγ1 (PLCγ1), Ca2+, or protein kinase C (PKC) impair clathrin-mediated endocytosis of EGFR, the formation of CCPs harboring EGFR, and EGFR signaling. Each of these manipulations was without effect on the clathrin-mediated endocytosis of transferrin receptor (TfR). EGFR and TfR were recruited to largely distinct clathrin structures. In addition to control of initiation and assembly of CCPs, EGF stimulation also elicited a Ca2+- and PKC-dependent reduction in synaptojanin1 recruitment to clathrin structures, indicating broad control of CCP assembly by Ca2+ signals. Hence EGFR elicits PLCγ1-calcium signals to facilitate formation of a subset of CCPs, thus modulating its own signaling and endocytosis. This provides evidence for the versatility of CCPs to control diverse cellular processes.

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.

Receptors, acceptors, and the action of polypeptide hormones: illustrative studies with epidermal growth factor (urogastrone)

1983 ◽  
Vol 61 (7) ◽  
pp. 670-682 ◽  
Author(s):  
M. O'Connor-McCourt ◽  
M. D. Hollenberg
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Density Lipoprotein ◽  
Peptide Hormone ◽  
Peptide Hormones ◽  
Hormone Action ◽  
Coated Pits ◽  
Polypeptide Hormones ◽  
Epidermal Growth

The action of polypeptide hormones is discussed in the context of observations made both with receptors, like the ones for epidermal growth factor (urogastrone) (EGF-URO), insulin, and gonadotropin-releasing hormone (GnRH), and with acceptors, like the ones that participate in the adsorptive pinocytosis of substances like cholesterol (low density lipoprotein acceptor), cobalamin (transcobalamin-II acceptor), and asialoglycoproteins (galactose acceptor). The overall processes that both pharmacologic receptors and acceptors undergo subsequent to ligand binding comprise (a) microclustering; (b) aggregation at both coated ("coated pits") and uncoated plasma membrane regions; (c) internalization, with the formation of endocytic vesicles (endosomes or receptosomes); and (d) either recycling of endosomes (receptosomes) to the plasma membrane or fusion of endosomes (receptosomes) with lysosomes, accompanied by proteolytic degradation of the internalized ligand–receptor complex. The relevance of these processes to hormone action is discussed. Further, illustrative studies with EGF-URO are used to highlight general mechanisms that are thought to participate in the action of a variety of peptide hormones. The microclustering event and the concomitant receptor-regulated membrane phosphorylation reactions are emphasized as key plasma-membrane-localized processes that are very likely involved in the rapid (minutes to tens of minutes) actions of many peptide hormones. For the delayed actions of hormones (hours to tens of hours; e.g., DNA synthesis), the potential importance of nonlysosomal receptor processing, mediated by SH-requiring proteases that generate intracellular receptor fragments, is pointed out. A scheme for the action of EGF-URO is outlined and a model for peptide hormone action is presented that illustrates how multiple chemical mediators of hormone action may result from the combination of a single hormone with its unique receptor.

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