EGF-Receptor Signaling in Endocytosis Deficient Cells

Award DAMDl7-99-l-9367 seeks to understand the role of membrane trafficking in Epidermal Growth Factor Receptor EGFR signal transduction. We have used a tissue culture model system HeLa cells to isolate the activated EGER at a distinct stage in the endocytic pathway, namely the early endosome. Stable expression of a constitutively active form of the small molecular weight guanine nucleotide binding protein rab5, we recapitulated a previously described phenotype of enlarged, functional endosomes. When we biochemically explored the consequence of the enlarged endosome, we found unaltered EGFR trafficking though the endocytic pathway. A more thoroughly examination revealed that the originally reported accumulation of transferring receptors in the early endosomal compartment was not occurring. We next did a series of detailed studies using a transient expression system in HeLa cells in which the level and time of constitutively active rab5 overexpression were varied. From these studies, we discovered that while under virtually all conditions we could obtain the previously described enlarged endosomal phenotype, under no conditions did we observe changes in membrane trafficking. Thus, we conclude that changes in endosomal morphology i.e. enlarged endosomes in HeLa cells do not accurately forecast changes in transferring receptor or EGFR membrane trafficking.