Comparison of the methods for determining cell-surface and intracellular receptors for epidermal growth factor in the rat liver.
We compared methods for determining the distribution of epidermal growth factor (EGF) receptors between the cell surface and the cell interior in the rat liver. Incubation of isolated hepatocytes with 100 nM EGF for 20 min at 37 degrees C remarkably decreased the cell-surface EGF receptor density (internalization of receptors). The detergent Brij 35 was previously reported to permit assay of the intracellular latent EGF receptors in liver homogenates, but in the present investigation, Brij 35 lowered the affinity of EGF for the receptor depending on the detergent concentration, and the appearance of latent receptors was not observed. In contrast, permeabilization of the cells with digitonin, followed by an acid-washing procedure, increased the EGF binding capacity to close to the control level. Hence, the EGF receptors, internalized together with EGF molecules, were not degraded for at least 20 min, and the digitonin method is suitable for quantifying the intracellular EGF receptors. The binding capacities of the digitonin-treated and untreated control cells showed no difference upon digitonin treatment, suggesting that the bulk of EGF receptors exists on the cell surface. Further, cell-surface EGF receptor density was determined after the i.v. administration of EGF (300 micrograms/kg) to rats. Isolated hepatocytes prepared 30 min after the administration of EGF showed little binding for EGF on the cell surface, while the cell-surface EGF receptor density recovered to close to control values in cells prepared after 3 hr.
Yanai
S
S
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Sugiyama
Y
Y
Iga
T
T
Fuwa
T
T
Hanano
M
M
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D004072:Digitonin; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D066246:ErbB Receptors / Q000032:analysis* / Q000187:drug effects; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D004072:Digitonin