This proposal is focused on determining the mechanisms and interactions required for the differential localization of homologous members of the P-type ATPase family. Work outlined in this proposal stems from the observation that Na/K-ATPase is restricted to the basolateral surface of most epithelial cell types while the H/K-ATPase is present on apical surface of these cells. In addition, the H/K-ATPase also appears to be uniquely associated with a subapical population storage vesicles. The presence of this molecule in a storage compartment appears to be responsible for the stimulated secretion of gastric acid following the fusion of these vesicles with apical cell membranes while the sensation of acid secretion is associated with retrieval of the molecule from the cell surface. Results generated in the previous funding period have demonstrated that differential localization of each of the a subunits of these proteins appears to be determined by sequences found in each of the fourth transmembrane spanning sequences (TM4). In addition, studies in transgenic mice appear to have defined a tyrosine-based sorting signal in the cytoplasmic tail of the H/K-ATPase which is required for reinternalization of this pump following stimulation of gastric parietal cells. These studies establish the fact that the sorting signals embedded within both subunits play critical roles in regulating their trafficking. In the next period of funding, the sorting signals embedded in the H/K-ATPase and Na/K-ATPase a subunit will be further characterized following expression cells of molecules containing alterations in TM4. Initially, chimeras will be generated in which specific portions of TM4 of each protein are interchanged and, following expression in cultured polarized epithelial cells, the fate of chimeric molecules determined by immunoflorescence and surface biotinylation. Individual residues in each subdomain of the TM4 necessary for a proper sorting of each molecule will then be assessed by site-directed mutagenesis. Biochemical characterization of chimeras in which TM4 from the H/K-ATPase has been incorporated into the Na/K-ATPase show that the resulting protein is apparently able to transport both protons and sodium. It seems likely then that some or all of the residues in TM4 account for the proton transport specificity of the H/K-ATPase. To further define this property within the H/K-ATPase TM4 domain, all molecules developed for targeting studies will also be assessed for their role in determining the number and nature of actions transported by each pump. Tracer flux assays, intracellular pH measurements, ATPase assays, and electrophysiological assays of pump driven current will be used to assess the biochemical characteristics of each molecule. Experiments will also test whether the TM4 is sufficient to target molecules to either apical or basolateral surfaces when incorporated within the context of foreign molecules. Finally, similar experimental paradigms will be used to define sequences within the b subunit which are involved in the vectoral targeting and endocytosis of the H/K-ATPase.
The second aim will investigate the molecular interactions defined by the TM4 domain of the a subunit and the tyrosine-based motif in the b subunit which are required for the proper molecular sorting of these molecules. In the case of TM4, experiments will focus on determining whether the sorting signal embedded in this domain interacts with specific lipids or proteins and subsequently determining whether these interactions are required for appropriate targeting. Parietal cell proteins which are responsible for mediating regulated internalization of the H/K-ATPase b subunit through interaction with tyrosine-based signals in the cytoplasmic tail will be identified through standard yeast two hybrid screens. Finally, the last aim will use a transgenic mouse model to determine whether domains in the H/K-ATPase a and b subunits responsible for appropriate targeting and recycling of this molecules are also acted upon in the context of the complex cell type in which they serve their critical physiological functions. Here, chimeric constructs created in relation to the goals of earlier specific aims will be expressed in transgenic mice lacking in endogenous H/K-ATPase and the localization, function, and pathology-associated mislocalization or misregulation of the chimeric proteins assessed in gastric parietal cells.
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