The binding and subsequent internalization of 125I-labelled transferrin has been studied in the human choriocarcinoma cell line, JAR. At 4°C, binding was time- and concentration-dependent and exhibited saturation kinetics. The results indicate 1.6 × 106 binding sites per cell surface with a Kd of 4.12 × 10-9 m. Experiments with saponin-permeabilized cells revealed a large intracellular receptor pool constituting 72 per cent of the total cell receptor population. Evidence of ligand internalization at 37°C was obtained by showing the progressive resistance of cell-associated radioactivity to acid treatment. Pulse-chase experiments showed that after internalization, ligand resided within the cell for about 6 min before being released back to the medium in an intact form. Ligand release, but not internalization, was almost completely inhibited by exposure to monensin. Preincubation of cells at 37°C in the presence of monensin and under transferrin-free conditions had no effect on the subsequent binding of 125I-transferrin. Pulse-chase experiments using 125I-labelled anti-transferrin receptor antibody suggest that receptors are internalized in the absence of added transferrin with a rate coefficient of 0.036 min-1. In the presence of unlabelled transferrin, the initial rate of labelled antibody internalization was increased (rate coefficient was 0.23 min-1) but the maximal amount of antibody internalized remained virtually unchanged. Internalized antibody accumulated intracellularly with no evidence of release or degradation.
ASJC Scopus subject areas
- Obstetrics and Gynecology