scholarly journals Transmodulation of the epidermal-growth-factor receptor in permeabilized 3T3 cells

1988 ◽  
Vol 256 (1) ◽  
pp. 109-115 ◽  
Author(s):  
F Walker ◽  
A W Burgess
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Egf Receptor ◽  
Fluid Phase ◽  
Egf Receptors ◽  
3T3 Cells ◽  
High Affinity ◽  
Kinase C ◽  
Fluid Phase Endocytosis ◽  
Epidermal Growth

Binding of murine epidermal growth factor (EGF) to its high-affinity receptor can be modulated by a variety of structurally unrelated mitogens. The transmodulation, however, is temperature-dependent and has not been observed in isolated membranes. We report here the transmodulation of high-affinity EGF receptors by platelet-derived growth factors (PDGF) and tumour-promoting phorbol esters in 3T3 cells even when they are rendered incapable of fluid-phase endocytosis by treatment with phenylarsine oxide or by permeabilization with lysophosphatidylcholine. The relative affinity of the EGF receptors in the absence of modulating agents is not significantly altered by phenylarsine oxide treatment. Thus the difference in affinity between the two classes of EGF receptors seems to be unrelated to dynamic membrane changes or to differential rates of internalization. In permeabilized cells, non-hydrolysable GTP analogues transmodulate the high-affinity EGF receptor; however, the effects of these analogues are blocked by the protein kinase C inhibitor chlorpromazine. In contrast, transmodulation by PDGF is not blocked by chloropromazine. Thus the high-affinity EGF receptor can be transmodulated by both protein kinase C-dependent or -independent pathways, and the transmodulation processes do not require fluid-phase endocytosis.

scholarly journals Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding.

1986 ◽  
Vol 102 (6) ◽  
pp. 2211-2222 ◽  
Author(s):  
I Zachary ◽  
J W Sinnett-Smith ◽  
E Rozengurt
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Cellular Protein ◽  
3T3 Cells ◽  
Kinase C ◽  
Epidermal Growth ◽  
Swiss 3T3

Addition of bombesin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an Mr 80,000 cellular protein (designated 80k). The effect was both concentration and time dependent; enhancement in 80k phosphorylation could be detected as early as 10 s after the addition of peptide. Recently, a rapid increase in the phosphorylation of an 80k cellular protein after treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact fibroblasts (Rozengurt, E., A. Rodriguez-Pena, and K. A. Smith, 1983, Proc. Natl. Acad. Sci. USA., 80:7244-7248; Rozengurt, E., A. Rodriguez-Pena, M. Coombs, and J. Sinnett-Smith, 1984, Proc. Natl. Acad. Sci. USA., 81:5748-5752). The 80k phosphoproteins generated in response to bombesin and to phorbol 12,13-dibutyrate were identical as judged by one- and two-dimensional PAGE and by peptide mapping after partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with phorbol 12,13-dibutyrate, which leads to the disappearance of protein kinase C activity, blocked the ability of bombesin to stimulate 80k. Bombesin also caused a rapid (1 min) inhibition of 125I-labeled epidermal growth factor (125I-EGF) binding to Swiss 3T3 cells. The inhibition was both concentration and temperature dependent and resulted from a marked decrease in the affinity of the EGF receptor for its ligand. Peptides structurally related to bombesin, including gastrin-releasing peptide, also stimulated 80k phosphorylation and inhibited 125I-EGF binding; both effects were selectively blocked by a novel bombesin antagonist. These results strongly suggest that these responses are mediated by specific high-affinity receptors that recognize the peptides of the bombesin family in Swiss 3T3 cells. While an increase in cytosolic Ca2+ concentration does not mediate the bombesin inhibition of 125I-EGF binding, the activation of protein kinase C in intact Swiss 3T3 cells by peptides of the bombesin family may lead to rapid inhibition of the binding of 125I-EGF to its cellular receptor.

scholarly journals Rapid stimulation of amyloid precursor protein release by epidermal growth factor: role of protein kinase C

1997 ◽  
Vol 327 (1) ◽  
pp. 245-249 ◽  
Author(s):  
Barbara E. SLACK ◽  
Jeffrey BREU ◽  
Lisa MUCHNICKI ◽  
Richard J. WURTMAN
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Amyloid Precursor Protein ◽  
Egf Receptor ◽  
A431 Cells ◽  
Kinase C ◽  
Epidermal Growth ◽  
Stimulation Of

The amyloid precursor protein (APP) of Alzheimer's disease is a transmembrane protein that is cleaved by an uncharacterized enzyme known as α-secretase within its extracellular/intraluminal domain after the activation of guanine nucleotide-binding protein-coupled receptors linked to phosphoinositide hydrolysis. The secretory process results in the release of large soluble derivatives of APP (APPs), and, when elicited by muscarinic receptor activation, exhibits both protein kinase C (PKC)-dependent and tyrosine phosphorylation-dependent components [Slack, Breu, Petryniak, Srivastava and Wurtman (1995) J. Biol. Chem. 270, 8337–8344]. In this report we examine the regulation of the release of APPs by epidermal growth factor (EGF) receptors, which possess intrinsic tyrosine kinase activity, and are coupled to a variety of effectors including phosphoinositide-specific phospholipase Cγ. In A431 cells, EGF caused time-dependent and dose-dependent increases in the formation of inositol phosphates in cultures prelabelled with myo-[3H]inositol, and in the release of APPs into the culture medium; the two responses exhibited similar time courses and EC50 values for EGF. Concomitant with these effects, there were concentration-dependent (3–300 ng/ml) increases in the phosphorylation of tyrosine residues in several proteins, including the EGF receptor itself. The specific PKC antagonist GF 109203X decreased the effect of EGF by approx. 35% at a concentration that abolished the stimulation of the release of APPs by the PKC activator PMA. Tyrphostin AG 1478, an inhibitor of EGF receptor tyrosine kinase, abolished the EGF-induced release of APPs. These results demonstrate that in A431 cells, activation of the EGF receptor stimulates α-secretase activity by a mechanism that is partly dependent on PKC activity.

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.

Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor

1988 ◽  
Vol 8 (6) ◽  
pp. 2302-2308
Author(s):  
E Livneh ◽  
T J Dull ◽  
E Berent ◽  
R Prywes ◽  
A Ullrich ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Egf Receptor ◽  
Wild Type ◽  
3T3 Cells ◽  
Kinase C ◽  
Epidermal Growth ◽  
Nih 3T3

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.

scholarly journals Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor.

1988 ◽  
Vol 8 (6) ◽  
pp. 2302-2308 ◽  
Author(s):  
E Livneh ◽  
T J Dull ◽  
E Berent ◽  
R Prywes ◽  
A Ullrich ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Egf Receptor ◽  
Wild Type ◽  
3T3 Cells ◽  
Kinase C ◽  
Epidermal Growth ◽  
Nih 3T3

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.

scholarly journals Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-ε

1993 ◽  
Vol 289 (3) ◽  
pp. 875-881 ◽  
Author(s):  
D Schaap ◽  
J van der Wal ◽  
W J van Blitterswijk ◽  
R L van der Bend ◽  
H L Ploegh
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Epidermal Growth

In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor.

Effects of phenothiazines on binding and processing of epidermal growth factor in 3T3 cells

1986 ◽  
Vol 251 (6) ◽  
pp. C904-C911 ◽  
Author(s):  
R. Selinfreund ◽  
P. H. Lin ◽  
J. L. Cooper ◽  
W. Wharton
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Rapid Decrease ◽  
3T3 Cells ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Rapid Reversal ◽  
Epidermal Growth

Chlorpromazine (CPZ) or the functionally related N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide caused a rapid decrease in binding of 125I-epidermal growth factor (EGF) that was due to a specific decrease in receptor affinity. The decrease in ligand binding was observed when cells were exposed to CPZ at either 4 degrees C or 37 degrees C but a rapid reversal of CPZs effects was observed only during a 37 degrees C incubation. In contrast to the decrease in 125I-EGF binding seen after short (30 min) accumulations at 37 degrees C, the presence of CPZ caused a large increase in the amount of cell-associated radioactivity after longer periods (over 1 h) of accumulation. Although the CPZ-induced effect was similar in extent to that observed after the addition of methylamine, the increased accumulation after CPZ was probably not due to a nonspecific ionic neutralization of the lysosomes. CPZ did not lower EGF binding in cultures chronically treated with a phorbol ester to reduce protein kinase C levels, although the CPZ-induced increases in accumulation were still observed in cells with reduced protein kinase C activity.

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