(52d) Analysis of Time Scales Involved in Ligand-Mediated Endocytosis of the EGF Receptor

Ligand-mediated endocytosis is an important cellular mechanism for regulating signaling initiated by the epidermal growth factor receptor (EGFR), a ubiquitously expressed receptor tyrosine kinase which plays critical roles in physiology and is frequently over-expressed in cancer.  The classical understanding of EGFR endocytosis holds that ligand binding promotes receptor kinase activity, phosphorylation of receptor cytoplasmic tyrosines, and recruitment of proteins involved in the formation of endocytic vesicles.  Recent work from our group has identified the presence of certain hidden time scales in this process which arise due to the reversibility of binding of proteins to phosphorylated EGFR tyrosines and the activity of protein tyrosine phosphatases at the plasma membrane.  In this presentation, we will describe our work developing a kinetic model of EGFR endocytosis which incorporates the processes of ligand binding, receptor dimerization and phosphorylation, receptor dephosphorylation by protein tyrosine phosphatases, binding of adapter proteins, recruitment into coated pits, and translocation of receptors from the plasma membrane to the cell interior.  Our model was constructed as a set of coupled ordinary differential equations describing the kinetics of these rate processes.  The model was regressed against quantitative experimental measurements of the kinetics of receptor phosphorylation, dephosphorylation, endocytosis, and binding of relevant intracellular adapter proteins including Grb2 and epsin.  All measurements were taken in cultured wild-type HeLa cells.  A variety of perturbations to the wild-type cellular environment were also explored in a HeLa cell background, including knockdown of specific receptor-like protein tyrosine phosphatases, expression of specific adapter proteins, and chemical inhibition of phosphatases and endocytosis.  After regression against experimental data, our model was able to explain the seemingly contradictory experimental findings that protein tyrosine phosphatases regulate EGFR phosphorylation at the plasma membrane but exert no control over EGFR endocytosis.  Given the known requirement of EGFR phosphorylation for EGFR endocytosis, a feature which is encoded in our model, the model was only able to explain these experimental observations through identification of a time scale for EGFR pit recruitment which was very small compared to that for receptor dephosphorylation.  This model prediction was validated by experimental assessment of the kinetics of binding of pit-associated adapter proteins to EGFR in response to EGF.  Thus, our study suggests that phosphatases act upon EGFR at the plasma membrane but only after EGFR recruitment to coated pits.  Our model further resolved that the rate limiting step in the ultimate translocation of EGFR to the cell interior is EGFR movement out of coated pits.  Our study represents a novel experimental and computational approach for understanding the complex process of ligand-mediated EGFR endocytosis within a framework which incorporates multiple competing rate processes occurring across multiple time scales.  The results of our analysis suggest interesting new possibilities for regulating EGFR endocytosis as a means to control EGFR-mediated signaling.