MECHANISMS OF ABERRANT EGF RECEPTOR SORTING IN POLYCYSTIC KIDNEY DISEASE

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Formation of epithelial cell polarity is a fundamental process in embryonic development and organogenesis. Polarized epithelia in adult animals form a physical barrier between the host and external environment essential for homeostasis. Polarized epithelia maintain distinct apical (Ap) and basolateral (BL) membrane domains, by actively regulating membrane distribution of lipids and proteins as well as the sub- membranous cytoskeleton unique to each surface. Many questions remain about how membrane polarity is achieved, chief among them how Ap and BL proteins are packaged in distinct transport vesicles at the trans-Golgi- network (TGN). Sorting of BL proteins is mediated in part through recognition of distinct cytoplasmic sorting signals that also appear to regulate polarized sorting in endosomes. Although many BL sorting signals have common features, consensus motifs have not emerged, making it unclear whether all BL signals mediate transport by the same or different pathways. We propose to address this question by studying a novel autonomous Bl signal which we have identified in the EGF receptor (EGFR). This signal is located between residues K652 to A674 in the EGFR juxta- membrane domain, and mediates BL transport of cytoplasmically truncated EGFRs and protein chimeras containing a luminal. This BL signal critical tyrosine and leucine residues and do not overlap any of the known EGFR endocytic signals, features that distinguish it from many other well- characterized BL sorting signals. Computer modeling suggests a propensity for this region to form an amphipathic helix, in contrast to other BL signals whose critical structure suggests a propensity for this region to form a amphipathic helix, in contrast to other BL signals whose critical structure in a beta-turn. This region also induce T654, a known substrate for protein kinase C, raising the possibility that its activity is regulated by phosphorylation. Immediate goals are to understand the mechanism by which this signal establishes and maintains EGFR' polarity. A long-term goal is to understand how genes which cause polycystic kidney disease alter this process, since non-polar EGFR expression is a common finding that renal cysts both in humans and animals disease models. The specific aims will: test the hypothesis that BL sorting of cytoplasmically truncated EGFRs is critically dependent on particular amino acids in the BL signal; test the hypothesis that residues K652 to A674 regulate BL transport of full-length EGFRs; test the hypothesis that residues K652 to A674 regulate polarized sorting in endosomes; and characterize elements of the EGFR sorting machinery by identifying proteins that interact with EGFR residues K652 to A674.