BINDING OF EPIDERMAL GROWTH-FACTOR (EGF) TO A CULTURED HUMAN GLIOMA CELL-LINE

We have studied binding of I-125-EGF to the human malignant glioma cell line U-343 MG aC12:6, which is planned to be used as a model system in studies of toxic effects of EGF conjugates. Special care has been taken to fulfil the requirements for a correct Scatchard analysis of binding parameters. Binding as a function of time, temperature and pH was investigated as well as dissociation and internalization of bound EGF. The stability of EGF during incubation was also determined. After binding to the receptor, EGF is rapidly internalized and degraded at physiological temperature. We found that binding experiments should be performed at 4-degrees-C, since at this temperature practically no internalization took place, whereas dissociation occurred. From displacement experiments using increasing concentrations of unlabelled EGF competing with I-125-EGF for binding, binding parameters were calculated using a computerized, nonlinear, least-squares regression analysis of binding data. We found that EGF bound to a class of high affinity receptors with an apparent dissociation constant K(D) of about 4 x 10(-10) M. The mean number of receptors was 25,000 per cell. In experiments where receptors were saturated with I-125-EGF an addititonal class of low affinity receptors was detected. This had an apparent K(D) of 1 x 10(-8) M with a mean receptor number per cell of 780,000. We also noticed enhanced dilution-induced dissociation of bound I-125-EGF in the presence of excess unlabelled EGF, suggesting negative cooperativity.