Digestive function in the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of a cAMP-dependent protein kinase (PKA) cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. This relocation of the H,K-ATPase occurs concomitantly with extensive remodeling of the actin cytoskeleton, which is also an essential step in the activation of acid secretion. While these aspects of parietal cell activation are well defined, the molecular mechanisms that couple PKA-mediated phosphorylation to mobilization of H,K-ATPases and cytoskeletal remodeling are not known. A candidate coupling protein, ezrin, was identified as an 80 kDa phosphoprotein in parietal cells whose phosphorylation by PKA was related to parietal cell activation. Binding of ezrin to actin filaments is essential for gastric acid secretion. However, little is known regarding the molecular mechanism(s) by which ezrin operates in gastric acid secretion. The long-term goal of our research is to delineate the role of ezrin in stimulus-coupled epithelial secretion. To address this question, three Specific Aims ale proposed: first, we will identify the regions of ezrin necessary for beta-actin association using epitope-tagging, chemical footprinting, and crosslinking approaches. These studies will involve a detailed analysis of the structural determinants that mediate a direct ezrin-actin contact. Binding domain data will be used to design peptides that potently and specifically perturb ezrin-actin interactions in in vitro binding assays. The function of this interaction will then be determined by the effects of the peptides on acid secretion using permeabilized gastric glands. Second, we plan to determine the role of protein phosphorylation in the regulation of ezrin function by first mapping PKA-mediated phosphorylation sites. The function of ezrin phosphorylation in acid secretion will then be defined by expressing non-phosphorylatable ezrin mutants in cultured parietal cells. Third, we will continue to identify and characterize novel ezrin-interacting proteins. These studies will be facilitated by using our newly generated monoclonal antibodies directed against a novel set of ezrin-binding proteins. Studying the molecular and cellular mechanisms underlying parietal cell activation is of general importance in understanding cellular physiology of regulated epithelial secretion in gut, and is also expected to be of great benefit in leading to pharmacological strategies for correcting abnormal gastric acid secretion in disorders such as gastric and duodenal ulcers, and gastroesophageal reflux disease.
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