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Binding of epidermal growth factor (EGF) to a cultured human glioma cell line
M Pråhl1, T Nederman, J Carlsson
1Department of Radiation Sciences, Uppsala University, Sweden.
Abstract:
We have studied binding of 125I-EGF to the human malignant glioma cell line U-343 MG aCl2:6, 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 125I-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 KD of about 4 x 10(-10) M. The mean number of receptors was 25,000 per cell. In experiments where receptors were saturated with 125I-EGF an additional class of low affinity receptors was detected. This had an apparent KD 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 125I-EGF in the presence of excess unlabelled EGF, suggesting negative cooperativity.
Insights
Epidermal growth factor (EGF) binds to high-affinity receptors on glioma cells, with optimal binding at 4°C to prevent internalization. This research provides crucial parameters for using EGF conjugates in toxicological studies.
Area of Science:
- Cell Biology
- Biochemistry
- Oncology
Background:
- The human malignant glioma cell line U-343 MG is a potential model for studying toxic effects of epidermal growth factor (EGF) conjugates.
- Accurate characterization of EGF receptor binding is essential for developing reliable experimental models.
Purpose of the Study:
- To investigate the binding kinetics and parameters of 125I-EGF to the U-343 MG human malignant glioma cell line.
- To determine optimal conditions for Scatchard analysis of EGF receptor binding and to characterize receptor affinity and number.
Main Methods:
- Scatchard analysis was employed to study 125I-EGF binding parameters.
- Binding experiments were conducted as a function of time, temperature, and pH.
- Dissociation, internalization, and EGF stability were assessed.
- Computerized, nonlinear, least-squares regression analysis was used to calculate binding parameters.
Main Results:
- EGF binding experiments are best performed at 4°C to minimize internalization and maximize dissociation.
- Two classes of EGF receptors were identified: high-affinity (KD ≈ 4 x 10⁻¹⁰ M, 25,000 receptors/cell) and low-affinity (KD ≈ 1 x 10⁻⁸ M, 780,000 receptors/cell).
- Enhanced dissociation of bound EGF was observed with excess unlabeled EGF, suggesting negative cooperativity.
Conclusions:
- The U-343 MG cell line exhibits distinct high- and low-affinity EGF receptors.
- Optimal conditions for studying EGF binding, including temperature, were established.
- Findings support the use of this cell line as a model for EGF conjugate toxicity studies, providing essential binding parameter data.