Epidermal growth factor receptor kinase translocation and activation in vivo.

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Abstract. EGF receptor internalization, recycling, and downregulation were evaluated in liver parenchyma as a function of increasing doses of injected EGE The effect of ligand occupancy in vivo on the kinetics and extent of internalization was studied with changes in the receptor content of isolated plasmalemma and endosome fractions evaluated by direct binding, Scatchard analysis, and Western blotting. For all doses of injected EGF, receptor was lost from the plasmalemma and accumulated in endosomes in a time-and dosedependent fashion. However, at doses of injected EGF equivalent to <50% surface receptor occupancy (i.e., <1 #g/100 g body weight), receptor levels returned by 120 min to initial values. This return was resistant to cycloheximide and therefore did not represent newly synthesized receptor. Neither was the return due to replenishment by an intracellular pool of low-affinity receptors as such a pool could not be detected by Scatchard analysis or Western blotting. Therefore, receptor return was due to the recycling of previously internalized receptor.At doses of injected EGF >50% receptor occupancy, net receptor loss-i.e., downregulation-was observed by evaluating the receptor content of total particulate fractions of liver homogenates. At the higher saturating doses of injected EGF (5 and 10 #g/100 g body weight), the majority of surface receptor content was lost by 15 min and remained low for at least an additional 105 min. As the kinetics of ligand clearance from the circulation and liver parenchyma were similar for all doses of EGF injected, then the ligandmediated regulation of surface receptor content and downregulation were not a result of a prolonged temporal interaction of ligand with receptor. Rather, the phenomena must be a consequence of the absolute concentrations of EGF interacting with receptor at the cell surface and/or in endosomes.

Section: Dose Response and Kinetics Of Translocation Of Binding Sites From The Cell Surface To Endosomesmentioning

confidence: 99%

“…Liver parenchyma of male rats is enriched in EGF receptors (2,14,15,19). Little is known of the relationship between surface recptor occupancy and the phenomenon of downregulation in an in vivo context.…”

mentioning

confidence: 99%

Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo.

Lai

Cameron

Wada

et al. 1989

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“…The 32p-labeling was done directly in the dishes, in a volume of 1.0 ml for 5 rain at 22°C. The final concentrations of buffer constituents were 25 mM Hepes, pH 7.0, 1 mM MgC12, 5 mM 7-32p-ATP, and 10 ng/ml EGE Phosphorylation was stopped by washing the cells twice with the 20 mM Hepes, pH 7.4, buffer containing the inhibitors, and the proteins then solubilized in 200 pi of 2× concentrated electrophoresis buffer (Kay et al, 1986;Rubin and Earp, 1983).…”

Section: ~2 P-protein Labelingmentioning

confidence: 99%

Epidermal growth factor-induced selective phosphorylation of cultured rat hepatocyte 55-kD cytokeratin before filament reorganization and DNA synthesis.

Baribault

Blouin

Bourgon

et al. 1989

Abstract. We have reported previously that the addition of dexamethasone to cultured quiescent suckling rat hepatocytes in the presence of insulin, a culture condition which does not cause growth activation, induces a selective increase in the synthesis of the 49-kD/55-kD cytokeratin (CK49/CK55) pair over a 24-h period. This increased synthesis coincides with the formation of dense filament networks reminiscent of those observed in situ at the cell periphery (Marceau, N., H. Baribault, and I. Leroux-Nicollet. 1985. Can. J. Biochem. Cell Biol. 63:448-457). We show here for the first time that when EGF is added 48 h after insulin and dexamethasone, there is an early preferential phosphorylation of the CK55 of the CK49/CK55 pair, an induced filament rearrangement from the cell periphery to the cytoplasm, and a subsequent entry into S phase and mitosis after a lag period of 8 h. Indirect immunofluorescence microscopy with monoclonal antibodies to CK49 and CK55 indicate that, while before EGF treatment the cytokeratin filaments were mainly distributed near the cell periphery, the addition of EGF resulted in their reorganization to a predominantly cytoplasmic localization within <3 h. Antitubulin and anti-actin antibodies showed no detectable alteration in the distribution of microtubules and microfilaments. Pulse-chase measurements with [35S]methionine showed no apparent change in the turnover of either CK49 or CK55 during the period that precedes the initiation of DNA synthesis. 32p-labeling in vivo followed by SDS-PAGE demonstrated that CK55 was phosphorylated at a much higher level than CK49 in nonstimulated hepatocytes, and that the addition of EGF resulted in a selective stimulation of 32p-CK55 labeling within <30 min. Comparative analyses by two-dimensional PAGE of [35S]methionine and 32p-labeled cytokeratins at various times after EGF stimulation demonstrated a rapid increase in a first phosphorylated form of CK55 and the appearance of a second phosphorylated form at 30 rain poststimulation. The changes in the relative proportion of nonphosphorylated and phosphorylated forms were confirmed by immunoblotting with the anti-CK55 monoclonal antibody. Determinations of the 32p-labeled phosphoamino acids of CK55 extracted from the gels demonstrated that the radioactivity was mostly in serine residues. Labeling of Triton-permeabilized hepatocytes with 3,32p-ATP after treatment with EGF for 30 min to 3 h at 37°C, also demonstrated a phosphorylation of CK55 and CK49 as well, implying that the EGF-responsive serine protein kinase is detergent insoluble and probably part of the surface membrane skeleton. We propose that early selective CK55 phosphorylation at serine residues and subsequent rearrangements of CK49/CK55 filaments from cell periphery to cytoplasm are EGF receptor driven cell surface reactions that are part of the cascade of events that culminate in the initiation of DNA synthesis and subsequent cell division of hepatocytes.

“…HE EGF receptor contains intrinsic tyrosine kinase activity which appears to be active after internalization into endosomes (12,20). EGF receptor tyrosine kinase activity has been postulated by Honegger et al (19) to control downregulation.…”

mentioning

confidence: 99%

Ligand-mediated autophosphorylation activity of the epidermal growth factor receptor during internalization.

Lai

Cameron

Doherty

et al. 1989

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Abstract. The association of EGF with its receptor in endosomeS isolated from rat liver homogenates was assessed biochemically by polyethylene glycol precipitation and morphologically by electron microscope radioautography. The proportion of receptor-bound ligand in endosomes at 15 min after the injection of doses of 0.1 and 1 #g EGF/100 g body weight was 57 %. This value increased to 77 % for the dose of 10 /xg EGF injected. Quantitative electron microscope radioautography carried out on endosomes isolated at 15 min after the injection of 10/~g J25I-EGF demonstrated that most radiolabel was over the endosomal periphery thereby indicating that ligand-receptor complexes were in the bounding membrane but not in intraluminal vesicles of the content. EGF receptor autophosphorylation activity during internalization was evaluated in plasmalemma and endosome fractions.This activity was markedly but transiently reduced on the cell surface shortly after the administration of saturating doses of EGE The same activity, however, was augmented and prolonged in endosomes for up to 30 min after EGF injection. The transient desensitization of cell surface activity was not due to prior in vivo phosphorylation since receptor dephosphorylation in vitro failed to restore autophosphorylation activity. Transient desensitization of cell surface autophosphorylation activity coincided with a diminished capacity for endocytosis of ~25I-EGF with endocytosis returning to normal after the restoration of cell surface autophosphorylation activity. The inhibition of cell surface autophosphorylation activity and the activation of endosomal autophosphorylation activity coincident with downregulation suggest that EGF receptor traffic is governed by ligand-regulated phosphorylation activity.T HE EGF receptor contains intrinsic tyrosine kinase activity which appears to be active after internalization into endosomes (12,20). EGF receptor tyrosine kinase activity has been postulated by Honegger et al. (19) to control downregulation. Substitution of the ATP binding Lys 721 of the receptor for Ala and subsequent transfection into receptor-deficient NIH 3T3 cells resulted in the absence of downregulation but the maintenance of receptor-mediated endocytosis of EGE Hence, constitutive receptor recycling was proposed by these authors (19). The role of the tyrosine kinase domain of the EGF receptor in regulating internalization and traffic of the receptor has also been suggested by the mutational analyses carried out by Lin et al. (30) The companion paper (29) demonstrated that downregulation of the EGF receptor in vivo was evident at saturating doses of injected ligand but not when receptor occupancy was ~<50%. Downregulation was speculated to be some consequence of the interaction of a threshold concentration of EGF and its receptor at the cell surface and/or in endosomes.Sorting of incoming receptors and ligands is regulated within the endosomal apparatus (6,21,33,40), and EGF receptor phosphorylation is ligand dependent. We have consequently examined t...