Regulated expression of growth factor receptor provides for sensitive responses to changes in growth factor concentration and for the attenuation of responses that is necessary to control signal strength. Precisely controlled vectorial movement in each vesicular compartment is required for normal responses essential for growth, development and specialized function. When receptor tyrosine kinases (RTK) such as EGFR or erbB2 are over-expressed or mutant in human cancer normal attenuation via down-regulation does not occur and excessive signalling of growth occurs. Differential sorting of receptors in vesicular compartments depends on primary sequence information in the cytoplasmic domain that is recognized by cellular machinery. We propose to use defined sequence information or codes of RTKs to characterize the proteins that regulate vesicular trafficking and function in particular steps: vectorial movement from endosome to lysosomes, endocytosis form the cell surface and movement from the trans Golgi compartment to the basolateral surface. The sorting nexin (SNX) family of proteins will be used to characterize biochemical mechanisms involved in sorting RTKs and vesicular proteins to lysosomes. We proposes to characterize the protein components of the SNX vesicular compartment and to isolate components of the endosome to lysosome targeting pathway that interact with various domains of SNX molecules. An in vitro assay of late endosome to lysosome fusion will be established to analyze the biochemical effects of SNX proteins on this reaction. Studies of biochemical mechanisms involved in ligand-induced endocytosis of RTKs will focus on proteins that recognize class II sequences. The function of Enigma and of a related protein ENH will be studied in relation to insulin receptors because the LIM3 domain of Enigma specifically recognizes the endocytic code of InsR and to the cytoskeleton because the PDZ of Enigma specifically interacts with beta Tropomyosin 1. Region III of the carboxy terminus of EGFR contains strong endocytic code information but does not interact in vivo with AP2. This region will be used to isolate and functionally characterize the recognition machinery for these sequences. Because Basolateral expression of EGFR in polarized epithelial cells also requires region III of the EGFR carboxy terminus the sequences necessary for this process will be compared to those required for endocytosis. Specifically in function will be correlated to molecular recognition of these sequences by cell proteins. These studies will provide information fundamental to understanding specificity in sorting cargo molecules in different vesicular compartments; proper vesicular sorting of the RTK class of cargo determines cell growth responses.
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