In response to an acid diet, the rate of H+ transport in the collecting tubule of the kidney is increased by changing the function of intercalated cells. Using a clonal cell line derived from beta cells (which secrete base by an apical CI:HCO3 exchanger), the applicant found that high seeding density converts them to an alpha type that secreted acid by a basolateral anion exchanger (kAEI) and an apical H-ATPase. kAEI, an alternately spliced form of the red cell band 3, gets re-targeted from apical to basolateral domains under the influence of a newly identified extracellular matrix (ECM) protein which the applicant has named hensin. Purified hensin converts beta to alpha types and it binds to a small (p27) protein. Signal transduction through the hensin pathway causes re-organization of the cytoskeleton. Using the yeast two hybrid system, the applicant identified a third protein in this pathway, kanadaptin, which binds to kAEI only in transport vesicles but not when kAEI has been retained in the basolateral membrane.
The aim of this proposal is to study the cell and molecular mechanism by which these three new proteins mediate the conversion of the epithelial polarity of these acid/base transporting cells. This process involves a change in phenotype of these cells and likely represents a critical step in differentiation of epithelia.
The first aim i s to complete the sequencing of hensin and to identify its mouse genomic sequence in preparation for knockout studies. To examine the mechanism by which it localizes in the ECM, the applicant will study its polymerization and fibrillar formation. The 27 kDa protein will be cloned using recently determined peptide sequences, and antibodies will be generated to examine the kinetics of its association with hensin. Identification of a p27/hensin receptor will then complete the extracellular limb of the hensin pathway. Using the SH3 binding domain of kanadaptin the applicant will identify the protein(s) that connects kAE I to the targeting machinery. Finally, the applicant will investigate the mechanism by which acid feeding might intersect with this signal transduction pathway. These studies into the mechanism by which epithelial cells target their proteins to their final destination will provide deeper insights into the processes that mediate epithelial differentiation, a process that is defective in many diseases including cancer.
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