[An immunocytochemical study of the spontaneous and ligand-induced endocytosis of EGF-receptor complexes in A431 cells].

The ligand-induced and spontaneous endocytosis of epidermal growth factor receptors in A431 cells were studied by immunocytochemical methods. In our experiments with 125I-EGF it was shown that the conjugates of monoclonal antibodies to EGF-receptor with 10 nm colloid gold particles (monoAB-gold) did not prevent from either binding of the ligand to EGF receptor, or internalization and recycling processes. A431 cells were incubated with monoAB-gold and 100 ng/ml EGF for 1 hour at 4 degrees C, were warmed up to 37 degrees C for 0, 1, 30 and 60 min, which was followed by fixation and embedding in Epon-Araldit for electron microscopy. We have shown labelling of the plasma membrane, coated pits, early and late endosomes, multivesicular bodies and lysosomes. The EGF-independent endocytosis of mono-AB-gold--EGF-R complexes was examined that may be considered as a constitutive endocytosis. The A431 cells were incubated with monoAB-gold for 1 hour at 4 degrees C and warmed up to 37 degrees C for 1 hour without EGF to be followed by the electron microscopic examination. Gold particles were observed associated to various regions of the plasma membrane, including coated and uncoated pits, and with various types of vesicles, including coated and uncoated vesicles, early and late endosomes, multivesicular bodies and lysosomes. The data obtained indicate that A431 cells have high levels of constitutive, spontaneous internalization. Estimation of the ligand-independent endocytosis rate is important for understanding the ligand-induced endocytosis and the role of internalization in the growth factor response. Some contradictory data discussed in the literature (Honegger et al., 1990; Felder et al., 1990; Wiley et al., 1991; Felder et al., 1992) can be explained by a supposition that the levels of spontaneous endocytosis in different cell lines may differ from one another, but that is not always estimated by the researchers.