Distinct saturable pathways for the endocytosis of different tyrosine motifs

Endocytosis of surface proteins through clathrin-coated pits required an internalization signal in the cytoplasmic domain. Two types of internalization signal have been described: one requiring a tyrosine as the critical residue (tyrosine-based motif), and the other consisting of either two consecutive leucines or an isoleucine and leucine (dileucine motif). Although it seems that these signals are necessary and sufficient for endocystic targeting, the mechanism of recognition is not well understood. To examine this question, tetracycline repressible cell lines were used to overexpression one of several receptors bearing a tyrosine-based internalization signal. By measuring the rates of endocytosis for either the overexpressed receptor, or that of other endogenous receptors, we were able to show that the endocytosis of identical receptors could be saturated, by a complete lack of competition exists between the transferrin receptor (TfR), the low-density lipoprotein receptor, and the epidermal growth factor receptor. Overexpression of any of these receptors resulted in its redistribution toward the cell surface, implying that entry into coated pits is limited. During high levels of TfR expression, however, a significant increase in the amount of surface Lamp1, but not low-density lipoprotein receptor, epidermal growth factor receptor, or Lamp2, is detected. This suggests that Lamp1 and TfR compete for the same enodcytic sites. Together, these results support the idea that there are at least three distinct saturable components involved in clathrin-mediated endocytosis.