The endocytotic rate constant. A cellular parameter for quantitating receptor-mediated endocytosis.

The endocytotic rate constant (k,) is a cellular constant which defines the probability of an occupied receptor being internalized in 1 min at 37 "C. We have derived an equation that expresses the relationship between k, and the relative amount of surface-bound and internalized ligand as a function of time. This equation was used as the basis for a graphical form of analysis called the In/Sur plot. This plot directly yields k, as well as the lag time that exists between ligand internalization and degradation. Accurate utilization of the In/Sur plot is dependent upon precise discrimination of surface-bound and internalized ligand, the existence of a lag time between ligand internalization and degradation, and a relatively constant amount of surface-bound ligand with respect to time. In studies utilizing '2SI-labeled epidermal growth factor (EGF) and human fibroblasts, we found that all three of the above conditions could be satisfied using physiological temperatures and ligand concentration that spanned the mitogenic range for this cell type. There was approximately a 16% chance that an occupied EGF receptor would be internalized in 1 min at 37 "C. After internalization, there was a delay of at least 15 min prior to the initiation of EGF degradation. The ke value for EGF receptors was a constant independent of the total number of occupied cell surface receptors, indicating that receptors cluster to independent internalization sites rather than to each other. We also found that it was possible to measure the effects of environmental perturbations such as temperature and pH on both endocytosis and ligand binding, thus allowing one to quantitate the role of these processes in the overall interaction of ligands with target cells.