The long-term objective of this project is to characterize the mechanism that mediates regulated, dynamic retention in endosomes. To accomplish this goal, we will characterize in Chinese hamster ovary (CHO) cells the endocytic behavior of vpTR, a chimera between the human transferrin receptor and IRAP (insulinregulated aminopeptidase). We have previously demonstrated that vpTR is trafficked by a specialized, insulin-regulated, dynamic retention mechanism in CHO cells.
The specific aims of this proposal are to characterize the motifs that determine dynamic retention within the endosomal system, to identify the proteins that interact with specialized targeting motifs, and to develop a model for the molecular mechanism of dynamic retention within the endosomes. To accomplish these objectives we will combine site-directed mutagenesis and NMR structure analysis to develop structure-function models for the motifs. These models will be tested by characterizing the trafficking of mutants expressed in CHO cells. The proteins that bind these motifs will be identified using various yeast-two hybrid methods for detecting protein-protein interactions. A variety of intact and semi-intact cell assays will be used to investigate the molecular mechanism of dynamic retention. Specialized and regulated trafficking processes are key for cell function and normal whole body physiology. Not surprisingly, it is becoming increasingly clear that perturbations in membrane trafficking are a common cause of disease in humans, and it is therefore important to gain an understanding of these processes at a molecular level. The importance of dynamic retention along the biosynthetic pathway is well appreciated, and it is likely that dynamic retention within endosomes is equally important. Analysis of vpTR trafficking in CHO cells provides us the opportunity to studying this mechanism in molecular detail. The insulin-regulated, dynamic retention mechanism in CHO cells is likely to be a more common mechanism than the specialized insulin-regulated trafficking in fat cells. Consequently the results of the studies in this proposal may have signficant impact on our understanding of membrane trafficking mechanisms in general as well as being instructive for future studies in fat and muscle cells.
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