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1 Molecular & Cell Biology, University of California, Berkeley, CA, USA
2 Pathology, University of Oklahoma Health Science Center, Oklahoma City, OK, USA
* To whom correspondence should be addressed. E-mail: jforte{at}berkeley.edu.
Ezrin is a member of ERM (ezrin, radixin, moesin) protein family that links F-actin to membranes. The N- and C-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 associated with cell activation. Ezrin-ezrin interactions are presumably involved in functional regulation of ezrin, thus became a subject of our study. Fluorescence resonance energy transfer (FRET) was examined with cyan (CFP) and yellow (YFP) fluorescent protein tagged ezrin incorporated into HeLa cells and primary cultures of parietal cells. Constructs included YFP at N-terminus of ezrin (YFP-Ez), CFP at C-terminus of ezrin (Ez-CFP), and doubly labeled ezrin, N-YFP-ezrin-CFP-C. FRET was probed by fluorescence microcopy and spectrofluorometry. Evidence of ezrin oligomer formation was provided by FRET in cells co-expressing Ez-CFP and YFP-Ez, and by co-immunoprecipitation of endogenous ezrin with FP-tagged ezrin. Thus intermolecular N-C binding in vivo is consistent with earlier in vitro studies. After separating ezrin oligomers from monomers, FRET was observed in both forms, indicating intramolecular and intermolecular N-C binding. When the distribution of native ezrin, as oligomers versus 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.
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