scholarly journals Receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes: receptor pathway.

1986 ◽  
Vol 102 (1) ◽  
pp. 24-36 ◽  
Author(s):  
W A Dunn ◽  
T P Connolly ◽  
A L Hubbard
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Rat Hepatocytes ◽  
Receptor Protein ◽  
Cell Periphery ◽  
Egf Receptors ◽  
Receptor Mediated Endocytosis ◽  
High Affinity ◽  
Epidermal Growth

Substantial amounts of epidermal growth factor (EGF) are cleared from the circulation by hepatocytes via receptor-mediated endocytosis and subsequently degraded within lysosomes. We have used a combined biochemical and morphological approach to examine the fate of the receptor after exposure to EGF. Polyclonal antibodies were prepared against the purified receptor and their specificity established by immunoprecipitation and immunoblotting techniques. The EGF receptor was then localized by immunofluorescence and immunoperoxidase techniques and quantified on immunoblots. In untreated livers, EGF receptor was restricted to the sinusoidal and lateral surfaces of hepatocytes. 2-4 min after exposure of cells to EGF, the receptor was found in small vesicles (i.e., coated vesicles) as well as larger vesicles and tubules at the cell periphery. By 15 min the receptor was found in multivesicular endosomes located near bile canaliculi. Exposure of hepatocytes to EGF also resulted in a rapid loss of receptor protein from total liver homogenates and a decrease in its half-life from 8.7 h in control livers to 2.5 h. This EGF-induced loss of receptors was not observed when lysosomal proteinases were inhibited by leupeptin or when endosome/lysosome fusion was prevented by low temperature (16 degrees C). In the presence of leupeptin, receptor could be detected in structures identified as lysosomes using acid-phosphatase cytochemistry. All these results suggested rapid internalization of EGF receptors in response to ligand and degradation within lysosomes. However, four times more ligand was degraded at 8 h than the number of high-affinity (Kd of 8-15 nM) EGF-binding sites lost, suggesting either (a) high-affinity receptors were recycled, and/or (b) more than 300,000 receptors were available for EGF uptake. We identified and characterized a latent pool of approximately 300,000 low-affinity receptors (Kd approximately 200 nM) that could be separated on sucrose gradients from the plasma membrane pool of approximately 300,000 high-affinity receptors (Kd of 8-15 nM). Despite the differences in their binding affinities, the high- and low-affinity receptors appeared to be structurally identical and were both EGF-dependent protein kinases. In addition, the dynamics of the low-affinity receptors were consistent with a functional role in EGF uptake and delivery to lysosomes.

Localization of epidermal growth factor receptors in the human prostate by biochemical and immunocytochemical methods

1987 ◽  
Vol 113 (1) ◽  
pp. 147-NP ◽  
Author(s):  
S. Q. Maddy ◽  
G. D. Chisholm ◽  
R. A. Hawkins ◽  
F. K. Habib
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Specific Binding ◽  
Receptor Protein ◽  
Immunocytochemical Staining ◽  
Egf Receptors ◽  
Cytosol Fraction ◽  
Variable Binding ◽  
Epidermal Growth

ABSTRACT The receptor for epidermal growth factor (EGF) was characterized in the particulate fraction from human benign prostatic hyperplasia (BPH) and was present in 85% of tissues analysed. The uptake of 125I-labelled EGF by BPH was dependent on both time and temperature, with maximum specific uptake achieved after incubation for 90 min at 37 °C. Binding characteristics revealed two classes of binding sites of higher (mean dissociation constant (Kd)±s.d.= 0·8 ± 0·2 nmol/l) and lower (Kd = 7·6 ±2·8 nmol/l) affinities. Competition studies demonstrated the specificity of the receptor assay since the binding of labelled EGF was abolished with excess unlabelled EGF but not with excess unlabelled human GH, human insulin, venom nerve growth factor, human FSH, human LH and human prolactin. There was a complex biphasic relationship between specific binding and protein concentration in the range 0·1–8 mg/ml. Subcellular fractionation of BPH homogenates demonstrated that the bulk of the specific binding was confined to the 800 g (crude heavy pellet) and 15 000 g (mitochondrial pellet) fractions. The 105 000 g (microsomal pellet) and the 105 000 g (cytosol fraction) exhibited low and variable binding capacities for the growth factor. The presence of EGF receptor was also confirmed by immunocytochemical staining of frozen sections from BPH using monoclonal antibody specific for EGF receptors. A positive correlation between 125I-labelled EGF binding and the intensity of staining was found. The presence of a specific EGF-binding receptor protein in human BPH tissues suggests that EGF may play a role in the pathogenesis of human BPH. J. Endocr. (1987) 113, 147–153

Menstrual cycle specific expression of epidermal growth factor receptors in human fallopian tube epithelium

1995 ◽  
Vol 147 (3) ◽  
pp. 553-563 ◽  
Author(s):  
K Adachi ◽  
H Kurachi ◽  
H Adachi ◽  
T Imai ◽  
M Sakata ◽  
...  
Keyword(s):  
Growth Factor ◽  
Menstrual Cycle ◽  
Epidermal Growth Factor ◽  
Plasma Membranes ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Receptor Protein ◽  
Egf Receptors ◽  
Specific Staining ◽  
Epidermal Growth

Abstract We studied the expression of epidermal growth factor (EGF) receptor protein and messenger RNA (mRNA) in human fallopian tubes at three stages of the menstrual cycle: early follicular (n=3), late follicular (n=3) and luteal (n=3). Immunohistochemical studies in the ampullary portion of the tubes showed that specific staining was localized to the epithelium and the vascular endothelium. Staining of the epithelium was intense at the late follicular and luteal stages, while it was weak at the early follicular stage. 125I-EGF binding study in the tubal plasma membranes revealed a class of high-affinity EGF receptors. Although dissociation constants were similar between the stages, numbers of binding sites at the late follicular and luteal stages were significantly (P<0·01) greater than those at the early follicular stage. Western blotting showed that tubal plasma membranes contain Mr 170 000 EGF receptor protein. The amounts were significantly (P<0·01, n=3) greater at the late follicular and luteal stages than those at the early follicular stage. Reverse transcription and polymerase chain reaction (RT-PCR) revealed that EGF receptor mRNA was expressed in all the 9 RNA samples (n=3 for each stage) from the tubal ampullary portion. The amounts were significantly (P<0·01, n=3) greater at the late follicular and luteal stages than those at the early follicular stage (by a competitive PCR). Increase in the amounts of EGF receptor protein and mRNA occurred in association with an increase in serum oestradiol but not progesterone levels. Next we examined whether EGF receptor and its ligands (EGF and transforming growth factor a) are directly induced by oestrogen. We found that specific staining for EGF receptor and its ligands in the tubal epithelium was detected (by immunohistochemistry) in postmenopausal women with oestrogen replacement (n = 3), but not in subjects without oestrogen replacement (n=3). These results suggested that EGF receptors in the human tubal epithelium are expressed in relation to specific stages of the menstrual cycle and that the expression may be induced by oestrogen. Journal of Endocrinology (1995) 147, 553–563

scholarly journals Inhibition of the apparent affinity of the epidermal growth factor receptor caused by phorbol diesters correlates with phosphorylation of threonine-654 but not other sites on the receptor

1986 ◽  
Vol 233 (2) ◽  
pp. 435-441 ◽  
Author(s):  
R J Davis ◽  
M P Czech
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Affinity Binding ◽  
Egf Receptors ◽  
A431 Cells ◽  
High Affinity Binding ◽  
Phosphorylation State ◽  
High Affinity ◽  
Epidermal Growth

Addition of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) to A431 human epidermoid carcinoma cells causes a marked increase in the phosphorylation state of the epidermal growth factor (EGF) receptor with a concomitant inhibition of both the high-affinity binding of 125I-EGF and the receptor tyrosine kinase activity. It was found in the present studies that the diuretic drug amiloride has no effect on the action of PMA to inhibit the binding of 125I-EGF. However, amiloride was observed to inhibit markedly the effect of PMA to cause a 3-fold increase in the phosphorylation state of the EGF receptors. In the presence of PMA and amiloride, the increase in the phosphorylation state of the EGF receptors was found to be only 1.2-fold over controls. Analysis of the EGF receptor phosphorylation sites by phosphopeptide mapping by reverse-phase h.p.l.c. demonstrated that PMA increases the phosphorylation state of the EGF receptor at many sites. One of these sites has been identified as a C-kinase substrate, threonine-654. In the presence of amiloride, PMA causes phosphorylation of threonine-654 to the same stoichiometry as that observed in the absence of amiloride. However, the marked increase in the phosphorylation state of the EGF receptor at other sites caused by PMA is abolished in the presence of amiloride. We conclude that the extensive phosphorylation of the EGF receptor at several sites caused by the addition of PMA to A431 cells is not required for the action of PMA to inhibit the high-affinity binding of 125I-EGF. The results indicate that the phosphorylation state of threonine-654 may play a role in this process.

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

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.

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

1986 ◽  
Vol 102 (2) ◽  
pp. 500-509 ◽  
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.

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

Reproduction ◽  
2005 ◽  
Vol 130 (4) ◽  
pp. 517-528 ◽  
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.

scholarly journals Perinuclear location and recycling of epidermal growth factor receptor kinase: immunofluorescent visualization using antibodies directed to kinase and extracellular domains.

1986 ◽  
Vol 103 (2) ◽  
pp. 333-342 ◽  
Author(s):  
U Murthy ◽  
M Basu ◽  
A Sen-Majumdar ◽  
M Das
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Protein Biosynthesis ◽  
Kinase Domain ◽  
Immunofluorescent Staining ◽  
Receptor Kinase ◽  
Egf Receptors ◽  
Epidermal Growth ◽  
Indirect Immunofluorescent

This paper describes studies on the migratory behavior of epidermal growth factor (EGF) receptor kinase using antibodies that are specific for either the kinase domain or the extracellular domain of the receptor. Antiserum was raised to a 42,000-D subfragment of EGF receptor, which was shown earlier to carry the kinase catalytic site but not the EGF-binding site. Another antiserum was raised to the pure intact 170,000-D EGF receptor. The specificities of these antibodies were established by immunoprecipitation and immunoblotting experiments. The domain specificity was examined by indirect immunofluorescent staining of fixed cells. The anti-42-kD peptide antibody could bind specifically to EGF receptors of both human and murine origin and was found to be directed to the cytoplasmic part of the molecule. It did not bind to EGF receptor-negative cells, which contained other types of tyrosine kinases. The antibodies raised against the intact receptor recognized only EGF receptor-specific epitopes and were directed to the extracellular part of the molecule. The anti-receptor antibodies described above were used to visualize the cyclic locomotory behavior of EGF receptor kinase under various conditions of EGF stimulation and withdrawal. The receptor was examined in fixed and permeabilized cells by indirect immunofluorescent staining. The results demonstrate the following: (a) the receptor kinase domain migrates to the perinuclear region upon challenge with EGF; (b) both extracellular and cytoplasmic domains of the receptor are involved in migration as a unit; (c) withdrawal of EGF results in rapid recycling of the perinuclear receptors to the plasma membrane; (d) this return to the cell surface is inhibited by methylamine, chloroquine, and monensin; and (e) neither the internal migration nor the recycling process is blocked by inhibitors of protein biosynthesis.

scholarly journals Sulphydryl agents modulate insulin- and epidermal growth factor (EGF)-receptor kinase via reaction with intracellular receptor domains: differential effects on basal versus activated receptors

1993 ◽  
Vol 292 (1) ◽  
pp. 217-223 ◽  
Author(s):  
S Clark ◽  
N Konstantopoulos
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Insulin Receptor ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Cytoplasmic Domain ◽  
Receptor Kinase ◽  
Egf Receptors ◽  
Receptor Structure ◽  
Epidermal Growth

Sulphydryl reagents have been shown to produce a variety of effects on insulin-receptor structure and function. However, localization of these effects to specific receptor domains has not been attempted. We have investigated this question with insulin- and epidermal growth factor (EGF)-receptors (both are receptor tyrosine kinases but have different sulphydryl/disulphide structures within the external domain), and the insulin receptor kinase (IRK) protein consisting solely of the insulin-receptor cytoplasmic domain and exhibiting constitutive kinase activity. Results showed a differential response between basal and activated receptors. The physiological reductant GSH stimulated basal receptor autophosphorylation, but was either without effect (EGF) or inhibited (insulin) activated receptors, and occurred without visible reduction of receptor structure. These results contrast with those obtained with dithiothreitol which appears to activate phosphorylation in association with reduction of the extracellular insulin-receptor disulphides, but is without effect on the EGF receptor or the IRK protein. Alkylating agents N-ethylmaleimide (NEM) and iodoacetamide (IAM) had opposing effects on receptor autophosphorylation. However, only in the basal state was IAM able to protect receptors from the inhibitory effect of NEM. Our results suggest that complex sulphydryl interactions can occur within the cytoplasmic domain of insulin- and EGF-receptors to alter receptor kinase activity. The basal and activated state of receptors is not the same with respect to sulphydryl reagent action, possibly due to conformational change in the receptor induced by ligand (insulin, EGF) or constitutive (IRK) activation.

Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors

1992 ◽  
Vol 12 (3) ◽  
pp. 981-990
Author(s):  
P Hu ◽  
B Margolis ◽  
E Y Skolnik ◽  
R Lammers ◽  
A Ullrich ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Factor Receptors ◽  
Sh2 Domain ◽  
Platelet Derived Growth Factor ◽  
Egf Receptors ◽  
Binding Assays ◽  
Expression Library ◽  
Epidermal Growth

One of the immediate cellular responses to stimulation by various growth factors is the activation of a phosphatidylinositol (PI) 3-kinase. We recently cloned the 85-kDa subunit of PI 3-kinase (p85) from a lambda gt11 expression library, using the tyrosine-phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor as a probe (E. Y. Skolnik, B. Margolis, M. Mohammadi, E. Lowenstein, R. Fischer, A. Drepps, A. Ullrich, and J. Schlessinger, Cell 65:83-90, 1991). In this study, we have examined the association of p85 with EGF and platelet-derived growth factor (PDGF) receptors and the tyrosine phosphorylation of p85 in 3T3 (HER14) cells in response to EGF and PDGF treatment. Treatment of cells with EGF or PDGF markedly increased the amount of p85 associated with EGF and PDGF receptors. Binding assays with glutathione S-transferase (GST) fusion proteins demonstrated that either Src homology region 2 (SH2) domain of p85 is sufficient for binding to EGF and PDGF receptors and that receptor tyrosine autophosphorylation is required for binding. Binding of a GST fusion protein expressing the N-terminal SH2 domain of p85 (GST-N-SH2) to EGF and PDGF receptors was half-maximally inhibited by 2 and 24 mM phosphotyrosine (P-Tyr), respectively, suggesting that the N-SH2 domain interacts more stably with PDGF receptors than with EGF receptors. The amount of receptor-p85 complex detected in HER14 cells treated with EGF or PDGF. Growth factor treatment also increased the amount of p85 found in anti-PDGF-treated HER14 cells, suggesting that the vast majority of p85 in the anti-P-Tyr fraction is receptor associated but not phosphorylated on tyrosine residues. Only upon transient overexpression of p85 and PDGF receptor did p85 become tyrosine phosphorylated. These are consistent with the hypothesis that p85 functions as an adaptor molecule that targets PI 3-kinase to activated growth factor receptors.

Amplification of the structurally and functionally altered epidermal growth factor receptor gene (c-erbB) in human brain tumors

1988 ◽  
Vol 8 (4) ◽  
pp. 1816-1820
Author(s):  
H Yamazaki ◽  
Y Fukui ◽  
Y Ueyama ◽  
N Tamaoki ◽  
T Kawamoto ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Receptor Gene ◽  
Growth Factor Receptor ◽  
Egf Receptors ◽  
Tumor Cell Membrane ◽  
Human Glioblastoma ◽  
Human Glioblastoma Multiforme ◽  
Epidermal Growth

By using Southern blot analysis, we found that in two cases of human glioblastoma multiforme, cells carried amplified c-erbB genes which bore short deletion mutations within the ligand-binding domain of the epidermal growth factor (EGF) receptor. The products of these mutated c-erbB genes were about 30 kilodalton (kDa) smaller than the normal 170-kDa EGF receptor, and the tumor cell membrane fractions containing the 140-kDa abnormal EGF receptor showed a significant elevation of tyrosine kinase activity without its ligand. In view of the similarity to the activated viral and cellular erbB genes in the avian system, these mutated and overexpressed EGF receptors might play a role in the onset or development of human glioblastoma cells.

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