The hepatic binding and uptake kinetics of epidermal growth factor: studies with isolated rat hepatocytes

Hepatocytes are known to bind and internalize a variety of small molecular weight proteins by a process known as receptor-mediated endocytosis (RME). The purpose of this investigation was to characterize the binding and uptake kinetics of a small protein known to be taken up by the liver by RME, epidermal growth factor (EGF), using suspensions of freshly isolated rat hepatocytes. Rat hepatocytes accumulated 125I-EGF (90 pM) in a temperature-dependent fashion. Isolated hepatocytes incubated at 37 degrees C with 125I-EGF began to release a TCA-soluble radiolabeled material into the incubation medium with a lag period of 20 min. EGF uptake by isolated hepatocytes was linear for only 60 seconds and displayed saturation kinetics (apparent Km of 4 nM and a Vmax of 105 fM/min/10(6) cells). Hepatocytes incubated at 4 degrees C bound, but did not internalize, EGF. Under these conditions, EGF binding was saturable at concentrations above 8 nM. A Scatchard analysis revealed that the average number of receptors per hepatocyte was 7.7 X 10(4) with a dissociation constant of 2.6 nM. These data demonstrate that freshly isolated hepatocytes are capable of binding, internalizing and metabolizing EGF and thus are a good model to study RME of small molecular weight proteins.