Molecular mechanisms of Basolateral targeting in polarized epithelial cells

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DESCRIPTION (provided by applicant): Epithelial cells are polarized into distinct apical and basolateral plasma membrane domains. The apical membrane faces the outside of the body and the basolateral membrane is in contact with connective tissues. During the lifetime of any given epithelial cell it is crucial for organ function that this established polarity is maintained. For example, loss of polarity enables a cell to disintegrate from a monolayer, which is an early step in metastatic cancer. Apical and basolateral plasma membrane domains have distinct sets of lipids and plasma membrane receptors. To maintain this distinct distribution, the cells continuously need to sort newly synthesized proteins along the biosynthetic pathway and recycle internalized receptors to the correct membrane. Basolateral targeting often depends on targeting determinants in the cytoplasmic tail of a transmembrane protein and cytosolic adaptor molecules that recognize them and mediate sorting. We identified one major cytosolic component, the clathrin-adaptor complex AP-1B (Folsch et al., 1999). Clathrin adaptor complexes are tetraheteromers. Typically the medium subunit interacts directly with the sorting signals and is therefore of particular interest. The medium subunit of AP-1B, mu1B is exclusively expressed in epithelial tissues and directly interacts with basolateral cargo molecules (Folsch et al., 2001). Our work will focus on defining the molecular mechanisms of AP-1B function and its site(s) of action. Moreover, we will analyze how the membrane recruitment of AP-1B is regulated. Furthermore, we will investigate the molecular interactions leading to AP-1B mediated basolateral targeting. My laboratory will combine cell biological, biochemical and genetic approaches to resolve the following specific aims: 1) At which intracellular site(s) does AP-1B fulfill its sorting function? 2) How is AP-1B membrane recruitment and vesicle formation regulated? 3) What is the molecular basis for AP-1B specific sorting events? A better understanding of the processes by which epithelial cells maintain polarity is essential, if we want to learn which defects in molecular sorting may transform epithelial cells into cancer cells, to name only one example of diseases associated with the loss of cell polarity.