Rate constants for binding, dissociation, and internalization of EGF: effect of receptor occupancy and ligand concentration.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Waters
C M
CM
Department of Biomedical Engineering, Vanderbilt University, Nashville, Tennessee 37235.
Oberg
K C
KC
Carpenter
G
G
Overholser
K A
KA
D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D005333:Fetus; D007700:Kinetics; D008024:Ligands; D008168:Lung / Q000378:metabolism*; D008433:Mathematics; D008954:Models, Biological; D011485:Protein Binding; D051381:Rats
D008024:Ligands; D004815:Epidermal Growth Factor; D066246:ErbB Receptors