Kinetics of epidermal growth factor binding and processing in isolated intact rat hepatocytes. Dynamic externalization of receptors during ligand internalization.
The kinetics of binding and processing of epidermal growth factor (EGF) was studied in freshly isolated rat hepatocytes. After isolation the hepatocytes had a nonhomogeneous population of surface EGF receptors consisting of approximately 9000 high-affinity sites (Kd 21 pM) and 165,000 low-affinity sites (Kd 0.62 nM). Incubation at 37 degrees C (45 min) increased the number of surface receptors per cell to about 260,000. This increase was selective for the low-affinity receptors and was cycloheximide-sensitive. During 5 h of incubation at 37 degrees C the hepatocytes internalized 6-7-times more EGF molecules than the number of cell surface receptors, based on clearance measurements. The uptake was unaffected by cycloheximide. Concomitant estimation, using acid/salt elution, of surface-bound EGF and internalized EGF showed that the number of internalized EGF molecules exceeded the decrease in surface-binding 6 times. The ratio between internalized EGF and the decrease in surface binding was temperature-dependent, being reduced to a one-to-one stoichiometry at 10 degrees C. After down-regulation (approximately equal to 75%) induced by 5 nM unlabeled EGF the surface EGF receptors did not recover during subsequent incubation (2 h) at 37 degrees C. However, the remaining surface receptors internalized EGF in ninefold excess of their number. The large discrepancy between internalization capacity and cell surface binding capacity was also found in the presence of cycloheximide. The results support the idea that internalized EGF receptors are partly replaced by externalization of preformed intracellular receptors during EGF uptake in isolated hepatocytes, involving recycling of a small population of EGF receptors and/or recruitment of unexposed, pre-existing receptors.
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D004815:Epidermal Growth Factor; D003513:Cycloheximide; D066246:ErbB Receptors