Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization

We have developed a quantitative method to evaluate the interaction between cell surface receptors and the endocytic apparatus. This method exploits occupancy-dependent changes in internalization rates that occur in cells expressing high numbers of receptors. We found that constitutive internalization of the transferrin receptor behaves as a simple, first order process that is unaltered by ligand. Internalization of the epidermal growth factor (EGF) receptor, however, behaves as a saturable, second order process that is induced by receptor occupancy. Internalization of EGF receptors occurs through at least two distinct pathways: a low capacity pathway that has a relatively high affinity for occupied receptors, and a low affinity pathway that has a much higher capacity. The high affinity pathway was observed in all cells having receptors with intrinsic tyrosine kinase activity. Mutant EGF receptors lacking kinase activity could not utilize the high affinity pathway and were internalized only through the low affinity one. Mutated receptors with decreased affinity for kinase substrates were also internalized at decreased rates through the high affinity, inducible pathway. In the case of vitellogenin receptors in Xenopus oocytes, occupied receptors competed more efficiently for internalization than empty ones. Insulin increased the endocytic capacity of oocytes for vitellogenin receptors. Similarly, serum increased the capacity of the inducible pathway for EGF receptors in mammalian cells. These data are consistent with a model of internalization in which occupied receptors bind to specific cellular components that mediate rapid internalization. Ligand-induced internalization results from an increase in the affinity of occupied receptors for the endocytic apparatus. Hormones can also indirectly regulate endocytosis by increasing the number of coated pits or their rate of internalization. The ability to dissect receptor-specific effects from cell-specific ones should be very useful in investigating the molecular mechanisms of receptor mediated endocytosis.