Insulin-EGF receptor chimerae mediate tyrosine transphosphorylation and serine/threonine phosphorylation of kinase-deficient EGF receptors

Animal cell viruses provide valuable model systems for studying many normal cellular processes, including membrane protein sorting. The focus of this study is an integral membrane protein encoded by the E3 transcription region of human adenoviruses called E3-13.7, which diverts recycling EGF receptors to lysosomes without increasing the rate of receptor internalization or intrinsic receptor tyrosine kinase activity. Although E3-13.7 can be found on the plasma membrane when it is overexpressed, its effect on EGF receptor trafficking suggests that the plasma membrane is not its primary site of action. Using cell fractionation and immunocytochemical experimental approaches, we now report that the viral protein is located predominantly in early endosomes and limiting membranes of endosome-to-lysosome transport intermediates called multivesicular endosomes. We also demonstrate that E3-13.7 physically associates with EGF receptors undergoing E3-13.7–mediated down-regulation in early endosomes. Receptor–viral protein complexes then dissociate, and EGF receptors proceed to lysosomes, where they are degraded, while E3-13.7 is retained in endosomes. We conclude that E3-13.7 is a resident early endocytic protein independent of EGF receptor expression, because it has identical intracellular localization in mouse cells lacking endogenous receptors and cells expressing a human cytomegalovirus-driven receptor cDNA. Finally, we demonstrate that EGF receptor residues 675–697 are required for E3-13.7–mediated down-regulation. Interestingly, this sequence includes a known EGF receptor leucine-based lysosomal sorting signal used during ligand-induced trafficking, which is also conserved in the viral protein. E3-13.7, therefore, provides a novel model system for determining the molecular basis of selective membrane protein transport in the endocytic pathway. Our studies also suggest new paradigms for understanding EGF receptor sorting in endosomes and adenovirus pathogenesis.

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Insulin-receptor phosphotyrosyl-protein phosphatases

Biochemical Journal ◽

10.1042/bj2560893 ◽

1988 ◽

Vol 256 (3) ◽

pp. 893-902 ◽

Cited By ~ 26

Author(s):

M J King ◽

G J Sale

Keyword(s):

Insulin Receptor ◽

Phosphatase Activity ◽

Rat Liver ◽

Protein Phosphatase ◽

Egf Receptor ◽

Soluble Fraction ◽

Protein Phosphatases ◽

Egf Receptors ◽

Cell Extracts ◽

Dependent Protein

Calmodulin-dependent protein phosphatase has been proposed to be an important phosphotyrosyl-protein phosphatase. The ability of the enzyme to attack autophosphorylated insulin receptor was examined and compared with the known ability of the enzyme to act on autophosphorylated epidermal-growth-factor (EGF) receptor. Purified calmodulin-dependent protein phosphatase was shown to catalyse the complete dephosphorylation of phosphotyrosyl-(insulin receptor). When compared at similar concentrations, 32P-labelled EGF receptor was dephosphorylated at greater than 3 times the rate of 32P-labelled insulin receptor; both dephosphorylations exhibited similar dependence on metal ions and calmodulin. Native phosphotyrosyl-protein phosphatases in cell extracts were also characterized. With rat liver, heart or brain, most (75%) of the native phosphatase activity against both 32P-labelled insulin and EGF receptors was recovered in the particulate fraction of the cell, with only 25% in the soluble fraction. This subcellular distribution contrasts with results of previous studies using artificial substrates, which found most of the phosphotyrosyl-protein phosphatase activity in the soluble fraction of the cell. Properties of particulate and soluble phosphatase activity against 32P-labelled insulin and EGF receptors are reported. The contribution of calmodulin-dependent protein phosphatase activity to phosphotyrosyl-protein phosphatase activity in cell fractions was determined by utilizing the unique metal-ion dependence of calmodulin-dependent protein phosphatase. Whereas Ni2+ (1 mM) markedly activated the calmodulin-dependent protein phosphatase, it was found to inhibit potently both particulate and soluble phosphotyrosyl-protein phosphatase activity. In fractions from rat liver, brain and heart, total phosphotyrosyl-protein phosphatase activity against both 32P-labelled receptors was inhibited by 99.5 +/- 6% (mean +/- S.E.M., 30 observations) by Ni2+. Results of Ni2+ inhibition studies were confirmed by other methods. It is concluded that in cell extracts phosphotyrosyl-protein phosphatases other than calmodulin-dependent protein phosphatase are the major phosphotyrosyl-(insulin receptor) and -(EGF receptor) phosphatases.

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Heartbroken is a specific downstream mediator of FGF receptor signalling in Drosophila

Development ◽

10.1242/dev.125.22.4379 ◽

1998 ◽

Vol 125 (22) ◽

pp. 4379-4389 ◽

Cited By ~ 22

Author(s):

A.M. Michelson ◽

S. Gisselbrecht ◽

E. Buff ◽

J.B. Skeath

Keyword(s):

Egf Receptor ◽

Genetic Interaction ◽

Egf Receptors ◽

Tracheal System ◽

Fgf Receptor ◽

Receptor Signalling ◽

Early Migration ◽

New Gene ◽

Developmental Responses ◽

Downstream Mediator

Drosophila possesses two FGF receptors which are encoded by the heartless and breathless genes. HEARTLESS is essential for early migration and patterning of the embryonic mesoderm, while BREATHLESS is required for proper branching of the tracheal system. We have identified a new gene, heartbroken, that participates in the signalling pathways of both FGF receptors. Mutations in heartbroken are associated with defects in the migration and later specification of mesodermal and tracheal cells. Genetic interaction and epistasis experiments indicate that heartbroken acts downstream of the two FGF receptors but either upstream of or parallel to RAS1. Furthermore, heartbroken is involved in both the HEARTLESS- and BREATHLESS-dependent activation of MAPK. In contrast, EGF receptor-dependent embryonic functions and MAPK activation are not perturbed in heartbroken mutant embryos. A strong heartbroken allele also suppresses the effects of hyperactivated FGF but not EGF receptors. Thus, heartbroken may contribute to the specificity of developmental responses elicited by FGF receptor signalling.

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Evidence for epidermal growth factor (EGF)-induced intermolecular autophosphorylation of the EGF receptors in living cells

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4035-4044.1990 ◽

1990 ◽

Vol 10 (8) ◽

pp. 4035-4044

Author(s):

A M Honegger ◽

A Schmidt ◽

A Ullrich ◽

J Schlessinger

Keyword(s):

Signal Transduction ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Phosphorylation ◽

Egf Receptor ◽

Living Cells ◽

Egf Receptors ◽

Receptor Molecule ◽

Epidermal Growth ◽

Negative Mutant

In response to epidermal growth factor (EGF) stimulation, the intrinsic protein tyrosine kinase of EGF receptor is activated, leading to tyrosine phosphorylation of several cellular substrate proteins, including the EGF receptor molecule itself. To test the mechanism of EGF receptor autophosphorylation in living cells, we established transfected cell lines coexpressing a kinase-negative point mutant of EGF receptor (K721A) with an active EGF receptor mutant lacking 63 amino acids from its carboxy terminus. The addition of EGF to these cells caused tyrosine phosphorylation of the kinase-negative mutant by the active receptor molecule, demonstrating EGF receptor cross-phosphorylation in living cells. After internalization the kinase-negative mutant and CD63 have separate trafficking pathways. This limits their association and the extent of cross-phosphorylation of K721A by CD63. The coexpression of the kinase-negative mutant together with active EGF receptors in the same cells suppressed the mitogenic response toward EGF as compared with that in cells that express active receptors alone. The presence of the kinase-negative mutant functions as a negative dominant mutation suppressing the response of active EGF receptors, probably by interfering with EGF-induced signal transduction. It appears, therefore, that crucial events of signal transduction occur before K721A and active EGF receptors are separated by their different endocytic itineraries.

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3981 Muscarinic and EGF Receptors Activate The EGF Receptor In Cultured Rat Conjunctival Goblet Cells To Increase Intracellular [Ca2+] And Stimulate Mucin Secretion

SciVee ◽

10.4016/40842.01 ◽

2012 ◽

Author(s):

Robin Hodges ◽

Richard Carozza ◽

Jeffrey Bair ◽

Dayu Li ◽

Marie Shatos ◽

...

Keyword(s):

Egf Receptor ◽

Goblet Cells ◽

Egf Receptors ◽

Mucin Secretion ◽

Conjunctival Goblet Cells

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EGF promotes gastric mucosal restitution by activating Na+/H+exchange of epithelial cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00150.2001 ◽

2002 ◽

Vol 282 (5) ◽

pp. G866-G876 ◽

Cited By ~ 24

Author(s):

Akinori Yanaka ◽

Hideo Suzuki ◽

Takeshi Shibahara ◽

Hirofumi Matsui ◽

Akira Nakahara ◽

...

Keyword(s):

Electrical Resistance ◽

Egf Receptor ◽

Mucous Cells ◽

Egf Receptors ◽

Intracellular Acidosis ◽

Receptor Antibody ◽

Epidermal Growth ◽

Triton X ◽

Stimulation Of

This study was conducted to determine whether the contributions of epidermal growth factor (EGF) to gastric mucosal restitution after injury are mediated by stimulation of Na+/H+exchangers in surface mucous cells (SMC). Intact sheets of guinea pig gastric mucosae were incubated in vitro. Intracellular pH (pHi) in SMC was measured fluorometrically, using 2′,7′- bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Restitution after Triton X-100-induced injury was evaluated by recovery of electrical resistance. At neutral luminal pH, exogenous EGF (ex-EGF) increased pHiand enhanced restitution in the absence but not in the presence of serosal HCO[Formula: see text]. During exposure to luminal acid, ex-EGF not only prevented intracellular acidosis but also promoted restitution. These effects of ex-EGF were blocked by serosal amiloride or anti-EGF-receptor antibody. In the absence of ex-EGF, restitution was inhibited by replacement of luminal and serosal solutions with fresh solutions and was blocked more completely by serosal anti-EGF-receptor antibody. These results suggest that both endogenous and ex-EGF contribute to restitution via basolateral EGF receptors, with effects mediated, at least in part, by stimulation of basolateral Na+/H+exchangers.

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Effects of EGF on alveolar epithelial junctional permeability and active sodium transport

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1996.270.4.l559 ◽

1996 ◽

Vol 270 (4) ◽

pp. L559-L565 ◽

Cited By ~ 18

Author(s):

Z. Borok ◽

A. Hami ◽

S. I. Danto ◽

R. L. Lubman ◽

K. J. Kim ◽

...

Keyword(s):

Sodium Transport ◽

Egf Receptor ◽

Alveolar Epithelial Cell ◽

Short Circuit ◽

Specific Inhibitor ◽

Egf Receptors ◽

Active Sodium ◽

Alveolar Epithelial ◽

Active Sodium Transport ◽

Junctional Permeability

We evaluated the effects of epidermal growth factor (EGF) on transepithelial resistance (Rt) and active ion transport by alveolar epithelial cell (AEC) monolayers on tissue culture-treated polycarbonate filters. Rat type II cells were cultured in completely defined serum-free medium (MDSF) or MDSF supplemented with EGF. The addition of EGF from either day 0 (chronic) or day 4 (subacute) resulted in significant increases in Rt and short-circuit current (ISC) on day 5. After subacute exposure, these effects were delayed in onset by 6-12 h and sustained for > 24 h. Basolateral (but not apical) EGF was responsible for these effects, which were prevented by preincubation with tyrphostin RG-50864, a reversible specific inhibitor of the EGF receptor tyrosine kinase. ISC decreased, with a sensitivity to apical inhibitors of sodium transport in the order benzamil > amiloride > 5-(N-ethyl-N-isopropyl) amiloride in MDSF +/- EGF, and was completely inhibited by the addition of basolateral ouabain. Net sodium flux and Na+, K+ -ATPase activity both increased approximately 50% in the presence of EGF. These results indicate that 1) EGF decreases tight junctional permeability and increases active sodium transport by AEC monolayers via basolaterally located EGF receptors, and 2) the pathways for AEC sodium entry and exit (+/- EGF) are apical high amiloride affinity sodium channels and basolateral sodium pumps.

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The metalloprotease Kuzbanian (ADAM10) mediates the transactivation of EGF receptor by G protein–coupled receptors

The Journal of Cell Biology ◽

10.1083/jcb.200112026 ◽

2002 ◽

Vol 158 (2) ◽

pp. 221-226 ◽

Cited By ~ 218

Author(s):

Yibing Yan ◽

Kyoko Shirakabe ◽

Zena Werb

Keyword(s):

Signaling Pathways ◽

G Protein ◽

Egf Receptor ◽

Critical Role ◽

G Protein Coupled Receptors ◽

Heparin Binding ◽

Egf Receptors ◽

Gpcr Activation ◽

G Protein Coupled ◽

Stimulation Of

Communication between different signaling pathways enables cells to coordinate the responses to diverse environmental signals. Activation of the transmembrane growth factor precursors plays a critical role in this communication and often involves metalloprotease-mediated proteolysis. Stimulation of G protein–coupled receptors (GPCR) transactivates the EGF receptors (EGFRs), which occurs via a metalloprotease-dependent cleavage of heparin-binding EGF (HB-EGF). However, the metalloprotease mediating the transactivation remains elusive. We show that the integral membrane metalloprotease Kuzbanian (KUZ; ADAM10), which controls Notch signaling in Drosophila, stimulates GPCR transactivation of EGFR. Upon stimulation of the bombesin receptors, KUZ increases the docking and activation of adaptors Src homology 2 domain–containing protein and Gab1 on the EGFR, and activation of Ras and Erk. In contrast, transfection of a protease domain–deleted KUZ, or blocking endogenous KUZ by morpholino antisense oligonucleotides, suppresses the transactivation. The effect of KUZ on shedding of HB-EGF and consequent transactivation of the EGFR depends on its metalloprotease activity. GPCR activation enhances the association of KUZ and its substrate HB-EGF with tetraspanin CD9. Thus, KUZ regulates the relay between the GPCR and EGFR signaling pathways.

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Localization of the epidermal growth factor (EGF) receptor within the endosome of EGF-stimulated epidermoid carcinoma (A431) cells.

The Journal of Cell Biology ◽

10.1083/jcb.102.2.500 ◽

1986 ◽

Vol 102 (2) ◽

pp. 500-509 ◽

Cited By ~ 91

Author(s):

K Miller ◽

J Beardmore ◽

H Kanety ◽

J Schlessinger ◽

C R Hopkins

Keyword(s):

Acid Phosphatase ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Epidermoid Carcinoma ◽

Egf Receptors ◽

A431 Cells ◽

Thin Sections ◽

Receptor Complexes ◽

Epidermal Growth

We have followed the internalization pathway of both epidermal growth factor (EGF) and its receptor in human epidermoid carcinoma (A431) cells. Using EGF conjugated with horseradish peroxidase and anti-receptor monoclonal antibodies (TL5 and EGFR1) coupled either directly or indirectly to colloidal gold we have identified an extensive elaboration of endosomal compartments, consisting of a peripheral branching network of tubular cisternae connected to vacuolar elements that contain small vesicles and a pericentriolar compartment consisting of a tubular cisternal network connected to multivesicular bodies. Immunocytochemistry on frozen thin sections using receptor-specific antibody-gold revealed that at 4 degrees C in the presence of EGF, receptors were mainly on the plasma membrane and, to a lesser extent, within some elements of both the peripheral and pericentriolar endosomal compartments. Upon warming to 37 degrees C there was an EGF-dependent redistribution of most binding sites, first to the peripheral endosome compartment and then to the pericentriolar compartment and lysosomes. Upon warming only to 20 degrees C the ligand-receptor complex accumulated in the pericentriolar compartment. Acid phosphatase cytochemistry identifies hydrolytic activity only within secondary lysosomes and trans cisternae of the Golgi stacks. Together these observations suggest that the prelysosomal endosome compartment extends to the pericentriolar complex and that the transfer of EGF receptor complexes to the acid phosphatase-positive lysosome involves a discontinuous, temperature-dependent step.

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Epidermal growth factor receptor downregulation in cultured bovine cumulus cells: reconstitution of calcium signaling and stimulated membrane permeabilization

Reproduction ◽

10.1530/rep.1.00420 ◽

2005 ◽

Vol 130 (4) ◽

pp. 517-528 ◽

Cited By ~ 5

Author(s):

Zhong Zhao ◽

Damien Garbett ◽

Julia L Hill ◽

David J Gross

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Cumulus Cell ◽

Growth Factor Receptor ◽

Cumulus Cells ◽

Membrane Permeabilization ◽

Egf Receptors ◽

Epidermal Growth

Cumulus cell–oocyte complexes (COCs), culturedin vitro, are competent for maturation and fertilization. Inclusion of epidermal growth factor (EGF) in the COC culture medium enhancesin vitromaturation and subsequent embryonic development. It has been shown that isolated COCs exposed to EGF respond with a prolonged and pulsatile release of Ca2+into the extra-cellular medium and that cumulus cells (CCs) of complexes exhibit both a slow rise in intracellular [Ca2+] ([Ca2+]i) and plasma membrane permeabilization in response to EGF. These unusual signaling responses were examined in isolated, cultured bovine CCs. Few individual CCs showed [Ca2+]iincreases; the lack of response was found to be due to decrease of expression of endogenous EGF receptors after dissociation. CCs transfected with a human EGF receptor–GFP fusion protein showed robust, prolonged, EGF-stimulated [Ca2+]ielevations characteristic of CC responses in intact COCs. Many CCs that responded to EGF stimulation with a [Ca2+]irise also released entrapped fura-2 dye at the peak of the [Ca2+]iresponse, suggesting that CC permeabilization and death follows activation of the EGF receptor. The [Ca2+]ielevation due to EGF stimulation and subsequent membrane permeabilization was shown to be mediated by the inositol triphosphate signaling pathway.

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