Rate Constants for Binding, Dissociation, and Internalization of EGF

Effect of Receptor Occupancy and Ligand Concentration

Christopher Waters, Kerby C. Oberg, Graham Carpenter, Knowles A. Overholser

Research output: Contribution to journalArticle

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Abstract

We measured the kinetic parameters for interaction of epidermal growth factor (EGF) with fetal rat lung (FRL) cells under two sets of experimental conditions and applied sensitivity analysis to see which parameters were well-defined. In the first set of experiments (method 1), the kinetics of internalization and dissociation of radiolabeled EGF were measured with a temperature-shift protocol in medium initially devoid of free ligand. The initial concentration of radiolabeled EGF bound to the cell surface corresponded to levels of receptor occupancy ranging from approximately 200 receptors per cell to approximately 18 000 receptors per cell, a level at which EGF binding approaches saturation. In the second set of experiments (method 2), carried out at a constant temperature, we began with no surface-bound or internalized ligand. The initial free ligand concentration was varied from 0.2 to 50 ng/mL. In both sets of experiments, we measured surface-bound, internalized, and free 125I-EGF as functions of time and evaluated the parameters of a mathematical model of endocytosis. Sensitivity analysis showed that three rate constants were well-defined in this combination of two experimental approaches: ke, the endocytic rate constant; Ka, the association rate constant; and kd, the dissociation rate constant. The endocytic parameter ke was found to be independent of initial surface receptor occupancy (method 1); there was some indication that it increased with initial free ligand concentration in method 2. Neither kd nor ka was found to change with extent of initial surface receptor occupancy or initial free ligand concentration, respectively, a finding of significance, since diffusion theory predicts these parameters will vary with surface receptor occupancy. In addition, the measurement of kd and Ka enabled us to calculate EGF receptor affinity at 37 °C over a range of ligand concentrations.

Original languageEnglish (US)
Pages (from-to)3563-3569
Number of pages7
JournalBiochemistry
Volume29
Issue number14
DOIs
StatePublished - Apr 1 1990

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Epidermal Growth Factor Receptor
Epidermal Growth Factor
Rate constants
Ligands
Sensitivity analysis
Temperature
Experiments
Endocytosis
Kinetic parameters
Rats
Theoretical Models
Association reactions
Mathematical models
Lung
Kinetics

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Rate Constants for Binding, Dissociation, and Internalization of EGF : Effect of Receptor Occupancy and Ligand Concentration. / Waters, Christopher; Oberg, Kerby C.; Carpenter, Graham; Overholser, Knowles A.

In: Biochemistry, Vol. 29, No. 14, 01.04.1990, p. 3563-3569.

Research output: Contribution to journalArticle

Waters, Christopher ; Oberg, Kerby C. ; Carpenter, Graham ; Overholser, Knowles A. / Rate Constants for Binding, Dissociation, and Internalization of EGF : Effect of Receptor Occupancy and Ligand Concentration. In: Biochemistry. 1990 ; Vol. 29, No. 14. pp. 3563-3569.
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