Functional state of the epidermal growth factor-receptor complexes during their internalization in A-431 cells.

Abstract

Functional state of internalized epidermal growth factor (EGF) receptor in A431 cells has been studied. The use of photoaffinity [12"I]EGF derivative allowed us to establish that inside the cell the EGF retains its connection with the receptor. With the help of polyclonal antibodies to phosphotyrosine, it has been shown that EGF-receptor complexes maintain their phosphorylated state during internalization. The internalized EGF receptor kinase as well as that localized in the plasma membrane appeared to be able to phosphorylate synthetic peptide substrate introduced into the cell.The epidermal growth factor (EGF) receptor is a 170-kilodalton (kDa) integral membrane glycoprotein containing cytosol-oriented tyrosine-specific kinase (for a review, see reference 18). EGF binding stimulates receptor tyrosine kinase, resulting in both receptor and substrate phosphorylation (for a review, see reference 10). There is a lot of data showing that receptor kinase activity plays an important role in the transmission of mitogenic signal (for a review, see references 20 and 25).After EGF binding, the ligand-receptor complexes are rapidly internalized; they are then delivered to various endosomal structures and, finally, degraded within lysosomes (8,9,12,21,22). The role of the internalization process in triggering the biochemical reactions leading to DNA synthesis so far remains obscure. To elucidate this question, the functional state of internalized EGF receptor should be determined.Whether the EGF receptors retain their functional activity during the translocation from plasma membrane into endosomal compartment is yet to be clarified. Thus, the presence of active EGF receptor in the fractions of intracellular components isolated from homogenates of A-431 cells (5) and rat liver cells (13) has been shown. On the other hand, from the results of experiments on intact A-431 cells, Sturani and co-workers concluded that internalized EGF receptors failed to maintain the phosphorylated state of tyrosine residues (24). In this study, we compared the functional state of internalized and membrane-localized EGF-receptor complexes by using two experimental approaches: (i) cell exposure to EGF at low temperature, which is known to completely block internalization (1) and (ii) removal of surface-bound EGF by cell treatment with weak acid buffer (50 mM sodium acetate [pH 4.5 to 5.0] containing 150 mM NaCl). To identify phosphotyrosine-containing proteins, we have used affinity-purified polyclonal antibody to phosphotyrosine (anti-P-Tyr). As has been established previously, the major phosphoprotein interacting with this antibody in the EGF-stimulated A-431 cells is the EGF receptor (17).Effect After that, the cells were solubilized and two equivalent portions of each extract were analyzed. One of them was immunoprecipitated with anti-P-Tyr, and then both the immunoprecipitate and the total extract were processed by electrophoresis. Radioactivity corresponding to 175-kDa bands and total cell-associated radioactivity were measured. The immunop...

Quantitative image analysis of cellular protein translocation induced by magnetic microspheres: Application to the EGF Receptor

“…In addition to the colocalization of molecules with microspheres, the disposition of the plasma membrane around the microspheres constitutes an important analytical parameter. Upon stimulation by growth factor, activated EGF receptors rapidly internalize (19, 20). Thus, for EGF‐coated microspheres, the microsphere internalization provides important evidence that signaling proceeds in the same or very similar manner as with the soluble ligand.…”

Coordinated Traffic of Grb2 and Ras during Epidermal Growth Factor Receptor Endocytosis Visualized in Living Cells

“…Several lines of evidence suggest that signals can be generated by endosomal receptors and that these receptors may be important for persistent signaling processes (Baass et al, 1995;Carpenter, 2000). Thus, despite the fact that the acidic pH of endosomes might be expected to cause ligand-receptor dissociation and deactivation of the receptor, it has been demonstrated that EGF-receptor complexes remain intact, dimerized, and phosphorylated during early stages of endocytosis (Cohen and Fava, 1985;Lai et al, 1989;Nesterov et al, 1990;Sorkin and Carpenter, 1991). Furthermore, the association of EGFR with effector proteins was demonstrated in endosomal fractions from rat liver (Di Gugliemo et al, 1994) and mammary cells (Burke et al, 2001).…”

Annexins and Membrane Fusion

“…This has also been shown for another growth factor receptor, colony stimulating factor 1 (Carlberg et al, 1991). The EGF receptor kinase is known to be active in the multivesicular body, as EGF stimulation causes a prolonged phosphorylation of the receptor (Nesterov et al, 1990), and it has been shown to be more highly phosphorylated in endosomes than at the plasma membrane (Wada et al, 1992). In multivesicular bodies incubated with [γ-32 P]ATP the two major proteins that are phosphorylated are annexin I and the receptor itself.…”