Comparison of epidermal growth factor (EGF) receptor-receptor interactions in intact A431 cells and isolated plasma membranes. Large scale receptor micro-aggregation is not detected during EGF-stimulated early events.
We have used resonance energy transfer to monitor epidermal growth factor (EGF) receptor micro-aggregation at the surface of intact human epidermoid carcinoma (A431) cells. EGF molecules labeled with fluorescein isothiocyanate and eosin isothiocyanate were demonstrated to bind tightly to cellsurface receptors, to elicit immediate changes in cytosolic free [Ca2+], and to undergo endocytosis. Under conditions which maintain the integrity of the cell, we observed no energy transfer between the donor fluorescein isothiocyanate-labeled EGF molecules and the acceptor eosin isothiocyanate-labeled growth factors bound to receptors. However, after disruption of cells by Dounce homogenization, a significant degree of energy transfer was observed (approximately 10-20%) with membranes, indicative of receptor aggregation. These results suggest that EGF does not cause micro-aggregation of the majority of its receptors on the surface of intact A431 cells within the time period of the early events associated with growth factor action. Moreover, it appears that the A431 cells contain some component which imparts a constraint on the ability of EGF receptors to aggregate, and that some of this component is lost upon the disruption of cells.
Carraway
K L
KL
Department of Biochemistry, Molecular, and Cell Biology, Cornell University, Ithaca, New York 14853-6401.
Cerione
R A
RA
D002118:Calcium / Q000378:metabolism; D004705:Endocytosis; D004735:Energy Transfer; D004815:Epidermal Growth Factor / Q000502:physiology*; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence; D011940:Receptor Aggregation; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D002118:Calcium