This application for renewal focuses on the role of small GTP binding proteins in the trafficking mechanisms in parietal cells. A number of gastrointestinal epithelial cells, including gastric parietal cells and chloride-secreting epithelial cells, have adopted a common mechanism for regulated delivery of pumps and channels to their apical membranes. In the non-stimulated or resting state, these molecules are sequestered in intracellular membrane vesicles which are located beneath the apical plasma membrane. Upon appropriate stimulation, pumps and channels are delivered to the apical surface through a vectorial fusion event. With cessation of the stimulatory signal, the transporters are retrieved through membrane reinternalization into a new population of intracellular fusion-ready vesicles. This regulated apical targeting appears to be a specialized elaboration of a general constitutive apical membrane recycling system present in most epithelial cells. Recent studies have identified three important aspects of apical recycling systems. First, the recycling system is comprised of an aggregation of vesicles located in the apical region of epithelial cells. Second, processing through the recycling system is dependent on the microtubule based cytoskeleton. Third, the recycling compartment is defined by the presence of members of the Rab11 subfamily of small GTP-binding proteins. The studies described in this project focus on the gastric parietal cell as a critical model of apical recycling. The parietal cell is an appropriate model to study apical trafficking, since it manifests a massive elaboration of the apical recycling system: the H/K-ATPase containing tubulovesicle system. The PI and his group have demonstrated that two Rab11 family members, Rab11a and Rab25, are components of the H/K-ATPase containing tubulovesicles. The PI, therefore, hypothesizes that Rab11a and Rab25 regulate the apical recycling of the H/K-ATPase in parietal cells. The PI will seek to define critical aspects of parietal cell apical cycling through three specific aims. The first specific aim will focus on purification and characterization of the Rab11-GTPase activator protein (GAP) as an immediate upstream regulator of Rab11a and Rab25. The second specific aim is designed to define upstream and downstream regulators of Rab11a and Rab25 through both chemical crosslinking of tubulovesicle proteins and screening of parietal cell cDNAs in a yeast 2-hybrid library. Studies described for the third specific aim will delineate the role of microtubules in regulating the initiation and termination of parietal cell acid secretion. These studies will define critical aspects of the regulatory machinery controlling apical recycling of tubulovesicle membranes and H/K-ATPase in parietal cells. Since Rab11a and Rab25 are components of general apical recycling systems, insights gained in these investigations may lead to more general concepts governing the control of epithelial cell apical trafficking.
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