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.
Highlights
The functions of an epithelium depend on the polarized organization of its individual epithelial cells
Because of its cytolocalization and observed stimulation-dependent phosphorylation, it was postulated that ezrin couples the activation of protein kinase A (PKA)[3] to the apical membrane remodeling associated with parietal cell secretion
Overexpression of ARF6Q67L, a mutant lacking GTP hydrolysis activity, in cultured gastric glands inhibits acid secretion. These results suggest that ARF6 regulates gastric acid secretion in parietal cells and that the GTP hydrolysis cycle of ARF6 is essential for the activation pathway
Summary
The functions of an epithelium depend on the polarized organization of its individual epithelial cells. Tamura et al (8) demonstrated that knockdown ezrin in stomachs to Ͻ5% of the wild-type levels results in severe achlorhydria In these parietal cells, H,KATPase-containing tubulovesicles failed to fuse with the apical membrane, suggesting an essential role of ezrin in tubulovesicle docking. Recent studies show that ARF6 is mainly located in the parietal cell within the gastric glands, and it relocates from the cytoplasm to the apical membrane of parietal cells upon stimulation of acid secretion (e.g. see Ref. 9). Our recent proteomic study has identified a novel ARF6 GTPase-activating protein ACAP4 essential for volatile membrane remodeling during cell migration (10). To delineate the molecular function of PKA-mediated phosphorylation of ezrin in parietal cell secretion, we took advantage of our recent development of Streptolysin O-permeabilized gastric glands and assessed the requirement of phosphorylation of ezrin in parietal cell activation by the addition of recombinant ezrin and its mutants. We propose that ACAP4-ezrin interaction provides a link between tubulovesicle trafficking and apical membrane reorganization in parietal cell activation
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