Functional Study Of Ezrin-ACAP4 Complex Underlying Regulated Exocytosis Dynamics

Cell plasticity and dynamics are the determinants underlying the formation and specification of tissues and organs,which is essential for animal development and health.Cellular polarity governs functional specificities of different domains of a given cell,which synthesizes the cellular plasticity and dynamics.Gastric parietal cells is a typical polarized epithelial cell responsible for HCl secretion and intrinsic factor release.Because of its great cytological features associated with secretion-coupled transformation stimulated by gastric hormones,gastric parietal cell becomes a unique model system to dissect molecular mechanisms underlying cAMP-regulated exocytosis.In turn,a better understanding of parietal cell physiology offers efficient strategies to minimize or eradicate gastrointestinal diseases associated with aberrant parietal cell function.Helicobacter pylori persistently colonize the human stomach and have been linked to atrophic gastritis and gastric carcinoma.Although it is well-known that H. pylori infection can result in hypochlorhydria,the molecular mechanisms underlying this phenomenon remain poorly understood.Most of formerly study focused on the immunoregulatory cells.Here we show that VacA permeabilizes the apical membrane of gastric parietal cells and induces hypochlorhydria.The functional consequences of VacA infection on parietal cell physiology were studied using freshly isolated rabbit gastric glands and cultured parietal cells.Secretory activity of parietal cells was judged by aminopyrine uptake assay and confocal microscopic examination.VacA permeabilization induces an influx of extracellular calcium followed by activation of the calcium dependent proteolytic enzyme calpainâ… and subsequent proteolysis of Ezrin at ⁴⁶⁹Mer-Thr⁴⁷⁰,which results in the liberation of ezrin from the apical membrane of the parietal cells.Ezrin provides a regulated linkage between membrane proteins aud the cortical cytoskeleton,and also participates in signal-transduction pathways.Ezrin is localized to the apical membrane of parietal cells and couples the PKA activation cascade to the regulated HCl secretion in gastric parietal cells.VacA treatment inhibits acid secretion by preventing the recruitment of H,K-ATPase-containing tubulovesicles to the apical membrane of gastric parietal cells.Electron microscopic examination revealed that VacA treatment disrupts the radial arrangement of actin filaments in apical microvilli due to the loss of Ezrin integrity in parietal cells.Significantly,expression of calpain-resistant Ezrin restored the functional activity of parietal cells in the presence of VacA.Proteolysis of Ezrin in VacA-infected parietal cell is a novel mechanism underlying H.pylori-induced inhibition of acid secretion.Our results indicate that VacA disrupts the apical membrane-cytoskeletal interactions in gastric parietal cells,and thereby causes hypochlorhydria.The second aspect of my research seeks to define the functional role of actin-based cytoskeleton dynamics underlying HCl secretion.Earlier experiments in our laboratory demonstrated that ezrin,a F-actin binding protein,provides a regulated linkage between cAMP-mediated signaling cascade and volatile membrane-cytoskeletal remodeling.To delineate the molecular mechanisms underlying parietal cell acid secretion,we have identified a new protein complex containing Ezrin-ACAP4-ARF6 responsible for volatile membrane-cytoskeletal reorganization.Using the gastric acid secretion as our model system,here we provide the first evidence that ezrin interacts with ACAP4 in PKA-mediated phosphorylation-dependent manner.ACAP4 resides on cytoplasmic membrane of resting parietal cells and relocates with H,K-ATPase tot he apical membrane upon histamine-stimulation.Our biochemical studies indicate that the N-terminal ACAP4 (1-400 aa)binds to phospho-mimicking mutant ezrin in vitro,suggesting a conformation-oriented interaction.Importantly,ezrin specifies the apical distribution of ACAP4 in secreting gastric parietal cells as either suppression of ezrin eliminates the apical localization of ACAP4.ACAP4 is an ARF6 GTPase-activating protein and binds directly to ARF6-GTP.Over-expressing GAP-deficient ACAP4 results in an inhibition of apical membrane-cytoskeletal remodeling and parietal cell secretion. Taken together,these results define a novel molecular mechanism linking ARF6-ACAP4-Ezrin interactions to polarized epithelial secretion.In sum,my research work has discovered a novel Ezrin-ACAP4-ARF6 protein network and elucidated its molecular function in gastric HCl section.My study on Helicobacter pylori has identified that VacA toxin disrupts the apical membrane-cytoskeleton via hydrolysis of ezrin.The information derived from this research will better our understanding of molecular physiology of parietal cell secretion which ultimately aid to minimize or perhaps eradicate the discomforts associated with gastrointestinal diseases of aberrant HCl secretion.