Molecular Mechanism Of Polarized Vesicular Trafficking Underlying Gastric Acid Secretion

Exocytosis is essential for many physiological activities including fertilization, immune response, cell signaling, cellular growth and polarization, the formation of extracellular matrix. However, the molecular mechisms underlying regulated exocytosis have remained elusive despite great progresses made over the last two decades.Gastric parietal cells are polarized epithelial cells in which hydrochloric acid secretion is triggered by paracrine, endocrine, and neurocrine pathways. Stimulation of acid secretion typically involves an initial elevation of intracellular calcium and cAMP followed by activation of a cAMP-dependent protein kinase cascade, which triggers the translocation and insertion of the proton pump enzyme, H, K-ATPase, into apical plasma membranes of parietal cells and results in an activation of the pump for acid secretion into the glandular lumen. After secretory stimuli are withdrawn, the H⁺, K⁺-ATPase is withdrawn back into the cytoplasmic compartment. The stimulation-mediated membrane transformation formulates the membrane recycling hypothesis of HC1 secretion. Therefore, acid secretion in parietal cells provides a perfect model system to study cAMP-regulated vesicular trafficking and its relationship to proton pump dynamics.Membrane-associated helical proteins known as soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) are crucial for vesicle fusion. In the brain, fusion of synaptic vesicles with the plasma membrane requires three SNARE proteins: syntaxin 1, SNAP25 (synaptosomal-associated protein 25 kDa) and synaptobrevin. The three SNARE proteins form a four-helical bundle—the SNARE complex that mediates membrane fusion. Our recent studies demonstrate the functional significance of membrane trafficking and fusion machinery components such as syntaxin 3, VAMP2, and SNAP25, in the parietal cell activation. In addition, we show the dynamic stimulation-associated redistribution of VAMP2 from H⁺, K⁺-ATPase-rich tubulovesicles to co-localize with SNAP-25 on the apical plasma membrane. Despite our demonstration of the functional importance of syntaxin 3 in parietal cell activation, it is still unclear how syntaxin 3 is involved in the tubulovesicular membrane dynamics triggered by histamine stimulation.In the present study, we have employed 2-dimention-electrophoresis and mass spectrometry to screen for phosphor-proteins required for live parietal cell activation, we identified the requirements of CDK5 and Munc18b in gastric acid secretion in parietal cells. Our pull-down assay revealed that Munc18b contains two binding sites for Stx3 which are localized to the N-terminal 156 amino acids and the most C-termianl 54 amino acids, respectively. The C-terminal Munc18 only bound Stx3 while the N-terminal Muncl8b bound to all syntaxin isoforms tested (stx1, 2, 3, and 4) equally. Further experiments in vitro and in vivo suggested the phoshorylation of Munc18b Thr 572 by CDK5 reduced its affinity for Stx3, however enhanced the complex of Munc18b with SNARE. Our studies reveal a novel regulatory mechanism underlying by which CDK5-mediated phosphorylation of Munc18b operates secretory vesicle docking and fusion in regulated exocytosis.Ezrin is a member of the ERM (ezrin/radixi/moesin) family which mediates the dynamic association between plasma membrane and actin cytoskeleton. In gastric parietal cell, it mainly localizes to the apical membrane, regulates the cell polarization and the localization of membrane proteins, furthermore, it plays an important role in the tubulovesicular membrane dynamics triggered by histamine stimulation. Since stx3 is an integral membrane protein involved in the epithelial cell polarization and mediates the fusion between tubulovesicle and apical membrane in parietal cells, then we presume that Stx3 may interact wih ezrin and this interaction may play an important role during the translocation of H⁺, K⁺-ATPase from plasma to the apical membrane in parietal cells.Our experiments in vitro verified our guess, and further assays revealed that Stx3-ezrin interaction was regulated by the phosphorylation of ezrin: when hatched Stx3 with various mutants of ezrin (ezrin^wt, ezrin^S66D, ezrin^T567D), it specifically interacted with ezrin^66D at physiological pH. Deletion mutants of Stx3 and ezrin were used to map the precise regions of Stx3-ezrin physical contacts. Our biochemical characterization demonstrated that the Habc domain of Stx3 and N-ERMAD domain of ezrin, suggesting that Ser66 phosphorylation-induced conformation change enables ezrin-Stx3 physical contact. To validate this hypothesis, we conducted atomic force microscopic analysis of recombinant ezrin proteins. Our analysis demonstrated Ser66 phosphorylation unfolds ezrin molecule to allow its N-terminus to bind Stx3. Taken together, our studies demonstrate an essential role of ezrin in the spatial control of polarized epithelial secretion.