Phospho-regulated ACAP4-Ezrin Interaction Is Essential for Histamine-stimulated Parietal Cell Secretion

Abstract

The ezrin-radixin-moesin proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to the regulated HCl secretion. Our recent proteomic study revealed a protein complex of ezrin-ACAP4-ARF6 essential for volatile membrane remodeling (Fang, Z., Miao, Y., Ding, X., Deng, H., Liu, S., Wang, F., Zhou, R., Watson, C., Fu, C., Hu, Q., Lillard, J. W., Jr., Powell, M., Chen, Y., Forte, J. G., and Yao, X. (2006) Mol. Cell Proteomics 5, 1437-1449). However, knowledge of whether ACAP4 physically interacts with ezrin and how their interaction is integrated into membrane-cytoskeletal remodeling has remained elusive. Here we provide the first evidence that ezrin interacts with ACAP4 in a protein kinase A-mediated phosphorylation-dependent manner through the N-terminal 400 amino acids of ACAP4. ACAP4 locates in the cytoplasmic membrane in resting parietal cells but translocates to the apical plasma membrane upon histamine stimulation. ACAP4 was precipitated with ezrin from secreting but not resting parietal cell lysates, suggesting a phospho-regulated interaction. Indeed, this interaction is abolished by phosphatase treatment and validated by an in vitro reconstitution assay using phospho-mimicking ezrin S66D . Importantly, ezrin specifies the apical distribution of ACAP4 in secreting parietal cells because either suppression of ezrin or overexpression of non-phosphorylatable ezrin prevents the apical localization of ACAP4. In addition, overexpressing GTPase-activating protein-deficient ACAP4 results in an inhibition of apical membrane-cytoskeletal remodeling and gastric acid secretion. Taken together, these results define a novel molecular mechanism linking ACAP4-ezrin interaction to polarized epithelial secretion.

Spatial Control of Proton Pump H,K-ATPase Docking at the Apical Membrane by Phosphorylation-coupled Ezrin-Syntaxin 3 Interaction

“…In addition, ezrin is essential for tubulovesicle docking during parietal cell secretion via phosphorylation of Ser-66 (22). To delineate the molecular mechanism underlying the Stx3-mediated recruitment of H,K-ATPase-containing membrane to the apical membrane during the parietal cell activation, we performed affinity purification of the ezrin complex from the apical membrane-rich fraction from histamine-stimulated parietal cells.…”

“…, and mCherry-ezrin S66D was described previously (11,12,22). The infection efficiency and level of various ezrin expression exhibit no difference.…”

“…The fusion proteins were then eluted in PBS containing 10 mM glutathione and then applied to a gel filtration column (Econ-Pac 10 DG; Bio-Rad) to remove glutathione and exchange PBS buffer for K ϩ buffer. Protein concentrations were determined by the Bradford assay (22).…”

Basics of gastric acid secretion

“…Точные механизмы, регу-лирующие встраивание Н+, К+-АТФазы в апикальную мембрану париетальной клетки, неизвестны. Имеются данные, что в этом процессе участвуют везикулярные белки (например, клатрин), актиновые микрофиламенты цитоскелета, актинсвязывающие белки (например, эзрин), а также небольшие G-белки семейства Rab (например, Rab10, Rab11, Rab25 и Rab27b) [15][16][17][18].…”

Immunohistochemical Analysis of the Acid Secretion Potency in Gastric Parietal Cells

“…Transformation between IC and TV occurs with redistribution of actin in the cell. Filamentous actins are anchored to the plasma membrane via phosphoryl ezrin, and most actin molecules are thought to form a globular structure in the inactive resting state, which molecules polymerize rapidly to form a filamentous structure upon active acid secretion [2, [6][7][8][9][10][11]. Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that is implicated in linking functional activities of the plasma membrane to the actin cytoskeleton.…”