Experimental and modeling studies of endosomal sorting using the epidermal growth factor/receptor system in fibroblasts

Receptor mediated endocytosis is the process by which cells internalize ligands which have specifically interacted with cell surface receptors. Cells have the ability to sort internalized ligands and their receptors to lysosomes for degradation, to recycle them back to the plasma membrane, or to sort ligands and receptors to separate destinations. We are interested in understanding how membrane-associated receptors are sorted within endosomes and how this process affects ligand sorting.The epidermal growth factor (EGF)/receptor system in fibroblasts has been studied as a model of the sorting of membrane-associated receptors and their ligands in non-polarized cells. We have focused on how receptor/ligand interactions influence sorting outcomes using a steady-state sorting assay with different members of the EGF family and several site-directed human EGF mutants in B82 fibroblasts. Mouse EGF, hEGF, and $\rm TGF\alpha$ all bind to EGF receptors (EGFR) with similar affinities at pH 7.4 and are internalized bound to EGFRs. These three ligands have different binding properties in the acidic endosomal environment and undergo distinct endosomal sorting outcomes. In addition, we observed that several site-directed human EGF mutants also exhibited altered sorting outcomes.We developed a mechanistic endosomal sorting model to help interpret the complicated experimental sorting results for the EGF/receptor system and to provide insight into the principles governing sorting. The model is based on the hypothesis that receptors may be selectively retained by the endosomal sorting apparatus and that this process may be modulated by receptor occupancy. The strength of this model lies in the ability of a single mechanism to account for a diverse range of sorting outcomes. The model predicts that the influence of selective endosomal retention of receptor/ligand complexes is seen in deviations of ligand sorting outcomes from fluid phase sorting behavior. Furthermore, the model suggests that selective endosomal retention of complexes within endosomes gives rise to three sorting regimes characterized by distinguishable qualitative trends in the dependence of ligand sorting fractions on intracellular ligand concentrations.A more complete understanding of how cellular and molecular properties govern endosomal sorting may allow this process to be manipulated in both biomedical and biotechnological applications.U of I OnlyETDs are only available to UIUC Users without author permissio