Ezrin oligomers are the membrane-bound dormant form in gastric parietal cells.
Ezrin is a member of ezrin, radixin, moesin (ERM) protein family that links F-actin to membranes. The NH(2)- and COOH-terminal association domains of ERM proteins, known respectively as N-ERMAD and C-ERMAD, participate in interactions with membrane proteins and F-actin, and intramolecular and intermolecular interactions within and among ERM proteins. In gastric parietal cells, ezrin is heavily represented on the apical membrane and is associated with cell activation. Ezrin-ezrin interactions are presumably involved in functional regulation of ezrin and thus became a subject of our study. Fluorescence resonance energy transfer (FRET) was examined with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged ezrin incorporated into HeLa cells and primary cultures of parietal cells. Constructs included YFP at the NH(2) terminus of ezrin (YFP-Ez), CFP at the COOH terminus of ezrin (Ez-CFP), and double-labeled ezrin (N-YFP-ezrin-CFP-C). FRET was probed using fluorescence microscopy and spectrofluorometry. Evidence of ezrin oligomer formation was found using FRET in cells coexpressing Ez-CFP and YFP-Ez and by performing coimmunoprecipitation of endogenous ezrin with fluorescent protein-tagged ezrin. Thus intermolecular NH(2)- and COOH-terminal association domain (N-C) binding in vivo is consistent with the findings of earlier in vitro studies. After the ezrin oligomers were separated from monomers, FRET was observed in both forms, indicating intramolecular and intermolecular N-C binding. When the distribution of native ezrin as oligomers vs. monomers was examined in resting and maximally stimulated parietal cells, a shift of ezrin oligomers to the monomeric form was correlated with stimulation, suggesting that ezrin oligomers are the membrane-bound dormant form in gastric parietal cells.
Zhu
Lixin
L
Department of Molecular and Cell Biology, University. of California, 245 Life Sciences Addition, MC 3200, Berkeley, CA 94720-3200, USA.
Liu
Yuechueng
Y
Forte
John G
JG
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D003598:Cytoskeletal Proteins; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D010295:Parietal Cells, Gastric / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D011817:Rabbits; D000099149:Ezrin
D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D000099149:Ezrin