Ontogeny and characterization of epidermal growth factor receptors on the fetal area of the sheep placenta

1993 ◽  
Vol 136 (1) ◽  
pp. 43-50 ◽  
Author(s):  
M. C. Lacroix ◽  
G. Kann
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Egf Receptors ◽  
High Affinity ◽  
Reducing Conditions ◽  
Sds Page ◽  
Ovine Placenta ◽  
Epidermal Growth

ABSTRACT Studies of the binding of 125I-labelled epidermal growth factor (EGF) to the ovine placenta were carried out on days 50, 90–100 and 140 of pregnancy. Membrane fractions were purified from the fetal area of the cotyledon. Two classes of binding sites were found. Their dissociation constants (Kd) were not significantly different for the three stages of pregnancy considered (high-affinity Kd 54–70·2 pmol/l; low-affinity Kd 12·2 to 19 nmol/l). However, the number of high-affinity binding sites on days 90–100 was significantly (P < 0·01) greater (146 ± 19 fmol/mg protein) than on either day 50 or day 140 (respectively 74·2 ± 1·26 and 56·3 ± 5·6 fmol/mg protein). Affinity cross-linking studies followed by SDS-PAGE under reducing conditions demonstrated that the major part of the EGF was specifically cross-linked to a protein of molecular weight of 150 kDa and to lesser extent to 180 kDa and 130 kDa proteins. Membranes prepared from unfrozen tissues, in the presence of sodium iodoacetate to reduce endogenous enzymatic conversion of the 180 kDa form to the 150 and 130 kDa forms, still exhibited a major EGF-binding protein of 150 kDa. The occurrence of an increased number of EGF receptors at the period of rapid cotyledonary growth which coincides with the increase in placental hormonal secretions suggests that EGF has a role in the development of the ovine placenta. Journal of Endocrinology (1993) 136, 43–50

Interaction of epidermal growth factor with receptors on human and porcine thyroid membranes

1984 ◽  
Vol 102 (1) ◽  
pp. 57-61 ◽  
Author(s):  
H. Humphries ◽  
S. MacNeil ◽  
D. S. Munro ◽  
S. Tomlinson
Keyword(s):  
Enzyme Activity ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Sites ◽  
Affinity Constant ◽  
Maximal Inhibition ◽  
High Affinity ◽  
Epidermal Growth ◽  
And Function ◽  
Bovine Tsh

ABSTRACT Recent evidence suggests that epidermal growth factor (EGF) may play an important role in the regulation of thyroid growth and function. We have examined the interaction of murine EGF (mEGF) with human and porcine thyroid membranes and compared this with the binding of bovine TSH (bTSH) using 125I-labelled hormones as tracers. The characteristics of the binding of mEGF were found to be similar for human and porcine thyroid membranes. Epidermal growth factor bound with high affinity (affinity constant = 1·4 × 109 l/mol); the density of binding sites was low compared with the TSH receptor. At 37 °C, the binding of 125I-labelled EGF was maximal at 1 h and was saturable in the presence of unlabelled EGF; half-maximal inhibition was at 1 ng EGF/tube (0·5 nmol/l) using 0·5 mg membrane protein/tube. Unlabelled bTSH had no effect on the binding of labelled EGF. Similarly, unlabelled EGF did not affect the binding of labelled TSH; hence it was concluded that mEGF and bTSH bound to independent sites. Epidermal growth factor had no effect on adenylate cyclase activity in membranes prepared from human non-toxic goitre; increasing concentrations of EGF did not affect basal, TSH-stimulated or fluoride-stimulated enzyme activity. J. Endocr. (1984) 102, 57–61

Characterization and autoradiographic localization of the epidermal growth factor receptor in the jejunum of neonatal and weaned pigs

1992 ◽  
Vol 4 (2) ◽  
pp. 183 ◽  
Author(s):  
D Kelly ◽  
M McFadyen ◽  
TP King ◽  
PJ Morgan
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Sites ◽  
Growth Factor Receptor ◽  
Saturation Curve ◽  
Egf Receptors ◽  
Weaned Pigs ◽  
Single Class ◽  
Epidermal Growth

Receptors for epidermal growth factor (EGF) were characterized on the intestinal membranes of newborn, sucking and weaned pigs. 125I-labelled EGF (125I-EGF) binding to membrane homogenates was time-dependent, saturable, linearly correlated to membrane protein and reversible. Analysis of saturation curve data revealed a single class of 125I-EGF binding sites in both newborn and weaned pigs. Receptor levels tended to be higher in weaned than in newborn pigs; the converse was true for the receptor affinity. In contrast, virtually no binding sites were found on the intestinal membranes of sucking pigs. Autoradiography in vitro of jejunal sections of newborn and weaned pigs demonstrated 125I-EGF receptors on both microvillar and basolateral surfaces of enterocytes, suggesting that luminal EGF could influence developmental processes in the intestine either directly or indirectly following transcytosis of the ligand.

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.

scholarly journals Characterization of a Drosophila protein that binds both epidermal growth factor and insulin-related growth factors.

1987 ◽  
Vol 105 (1) ◽  
pp. 449-456 ◽  
Author(s):  
J V Garcia ◽  
M P Stoppelli ◽  
K L Thompson ◽  
S J Decker ◽  
M R Rosner
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Dissociation Constants ◽  
Egf Receptors ◽  
Drosophila Protein ◽  
Epidermal Growth

The identification of a novel protein from Drosophila melanogaster that binds both mammalian epidermal growth factor (EGF) and insulin has been reported (Thompson, K. L., S. J. Decker, and M. R. Rosner, 1985, Proc. Natl. Acad. Sci. USA., 82:8443-8447). This 100-kD protein (designated dp100) is also recognized by an antiserum against the human EGF receptor. To further characterize the properties of this protein, we have determined the binding spectrum, glycosylation state, and cellular distribution of dp100. Our results indicate that dp100 binds to other insulin-like and EGF-like growth factors with dissociation constants ranging from 10(-6) to 10(-9) M, and these ligands compete with each other for binding to dp100. All other ligands tested, including platelet-derived growth factor, transforming growth factor-beta, nerve growth factor, and glucagon, either did not bind or bound with a Kd greater than 10(-6) M. Unlike the Drosophila insulin receptor, dp100 does not bind to wheat germ agglutinin and is present in a cytoplasmic as well as a membrane-bound form that cannot be differentiated by two-dimensional PAGE. Further, dp100 is the sole transforming growth factor-alpha-binding protein detected by affinity labeling in Drosophila Kc cells. These results indicate that dp100 shares properties in common with, but distinct from, the Drosophila homologues of the insulin and EGF receptors.

scholarly journals Cross talk among tyrosine kinase receptors in PC12 cells: desensitization of mitogenic epidermal growth factor receptors by the neurotrophic factors, nerve growth factor and basic fibroblast growth factor.

1993 ◽  
Vol 4 (7) ◽  
pp. 737-746 ◽  
Author(s):  
I Mothe ◽  
R Ballotti ◽  
S Tartare ◽  
A Kowalski-Chauvel ◽  
E Van Obberghen
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Fibroblast Growth Factor ◽  
Pc12 Cells ◽  
Binding Sites ◽  
Egf Receptor ◽  
Fibroblast Growth ◽  
High Affinity ◽  
Epidermal Growth

We have studied the effects of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) on epidermal growth factor (EGF) binding to PC12 cells. We show that NGF and bFGF rapidly induce a reduction in 125I-EGF binding to PC12 cells in a dose-dependent manner. This decrease amounts to 50% for NGF and 35% for bFGF. Both factors appear to act through a protein kinase C(PKC)-independent pathway, because their effect persists in PKC-downregulated PC12 cells. Scatchard analysis indicates that NGF and bFGF decrease the number of high affinity EGF binding sites. In addition to their effect on EGF binding, NGF and bFGF activate in intact PC12 cells one or several serine/threonine kinases leading to EGF receptor threonine phosphorylation. Using an in vitro phosphorylation system, we show that NGF- or bFGF-activated extracellular regulated kinase 1 (ERK1) is able to phosphorylate a kinase-deficient EGF receptor. Phosphoamino acid analysis indicates that this phosphorylation occurs mainly on threonine residues. Furthermore, two comparable phosphopeptides are observed in the EGF receptor, phosphorylated either in vivo after NGF treatment or in a cell-free system by NGF-activated ERK1. Finally, a good correlation was found between the time courses of ERK1 activation and 125I-EGF binding inhibition after NGF or bFGF treatment. In conclusion, in PC12 cells the NGF- and bFGF-stimulated ERK1 appears to be involved in the induction of the threonine phosphorylation of the EGF receptor and the decrease in the number of high affinity EGF binding sites.

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.

scholarly journals Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor.

1994 ◽  
Vol 14 (8) ◽  
pp. 5192-5201 ◽  
Author(s):  
A G Batzer ◽  
D Rotin ◽  
J M Ureña ◽  
E Y Skolnik ◽  
J Schlessinger
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Site ◽  
Binding Sites ◽  
Egf Receptor ◽  
Dissociation Constants ◽  
Interaction Analysis ◽  
Sh2 Domain ◽  
Epidermal Growth ◽  
Carboxy Terminal

We analyzed the binding site(s) for Grb2 on the epidermal growth factor (EGF) receptor (EGFR), using cell lines overexpressing EGFRs containing various point and deletion mutations in the carboxy-terminal tail. Results of co-immunoprecipitation experiments suggest that phosphotyrosines Y-1068 and Y-1173 mediate the binding of Grb2 to the EGFR. Competition experiments with synthetic phosphopeptides corresponding to known autophosphorylation sites on the EGFR demonstrated that phosphopeptides containing Y-1068, and to a lesser extent Y-1086, were able to inhibit the binding of Grb2 to the EGFR, while a Y-1173 peptide did not. These findings were confirmed by using a dephosphorylation protection assay and by measuring the dissociation constants of Grb2's SH2 domain to tyrosine-phosphorylated peptides, using real-time biospecific interaction analysis (BIAcore). From these studies, we concluded that Grb2 binds directly to the EGFR at Y-1068, to a lesser extent at Y-1086, and indirectly at Y-1173. Since Grb2 also binds Shc after EGF stimulation, we investigated whether Y-1173 is a binding site for the SH2 domain of Shc on the EGFR. Both competition experiments with synthetic phosphopeptides and dephosphorylation protection analysis demonstrated that Y-1173 and Y-992 are major and minor binding sites, respectively, for Shc on the EGFR. However, other phosphorylation sites in the carboxy-terminal tail of the EGFR are able to compensate for the loss of the main binding sites for Shc. These analyses reveal a hierarchy of interactions between Grb2 and Shc with the EGFR and indicate that Grb2 can bind the tyrosine-phosphorylated EGFR directly, as well as indirectly via Shc.

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