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

Abstract

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...

In vitro endosome‐lysosome transfer of dephosphorylated EGF receptor and Shc in rat liver

“…Other studies suggested that the tyrosine kinase itself plays a role in generating a lysosome targeting signal [27,28]. In accordance with this hypothesis, a greater and prolonged EGFR tyrosine phosphorylation has been demonstrated in rat liver endosomal membranes compared to the EGFR located at the plasma membrane [5,7,29,30]. The high phosphorylation content of internalized EGFR has been ascribed to a low dissociation and degradation state of EGF within the endosome [4,5,31].…”

Juxtacrine Activation of Epidermal Growth Factor (EGF) Receptor by Membrane-anchored Heparin-binding EGF-like Growth Factor Protects Epithelial Cells from Anoikis While Maintaining an Epithelial Phenotype

“…The fact that the membrane expression of EGFR in the MDCK 5aa cells was largely maintained even after 24 h of culture on polyHEMA (the time point with the highest EGFR activation) further supports juxtacrine activation of the EGFR. It is noteworthy that autocrine/paracrine activation of EGFR results in rapid receptor internalization and degradation (63).…”

Directing traffic in neural cells: determinants of receptor tyrosine kinase localization and cellular responses

“…In contrast, upstream components of ERK signaling, such as Ras and Shc, are accessible at the plasma membrane and endosomes (Di Guglielmo et al 1994;Haugh 2002). Although the ERK pathway may be activated globally, evidence suggests that the most potent activation of ERK occurs subsequent to internalization (Kay et al 1986;Lai et al 1989;Di Guglielmo et al 1994).…”