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J. Biol. Chem., Vol. 280, Issue 14, 13584-13592, April 8, 2005
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From the
School of Life Science, University of Science and Technology of China, Hefei 230027, Peoples Republic of China, the Department of Physiology, Morehouse School of Medicine, Atlanta, Georgia 30310, the ||Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, and the **State Key Laboratory of Cancer Biology, Xi'jing Hospital, Fourth Military Medical University, Xi'an, Shanxi 710032, People's Republic of China
Received for publication, October 20, 2004 , and in revised form, January 13, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-actin isoform in vivo (5) and preferentially bound to the -actin isoform in vitro (6). Because of its cytolocalization and observed stimulation-dependent phosphorylation, it was postulated that ezrin couples the activation of protein kinase A to the apical membrane remodeling associated with parietal cell secretion (4). Recently, we mapped the protein kinase A phosphorylation site on ezrin and illustrated the phosphoregulation of ezrin in gastric acid secretion (7). However, it is still not clear how ezrin links the apical membrane to the actin cytoskeleton during parietal cell activation.
ERM proteins all possess an 
300-residue N-terminal domain that shares sequence homology with the corresponding domain of erythrocyte band 4.1, followed by an 170-residue region predicted to be largely 
-helical and terminating in an 100-residue domain in which an F-actin-binding site resides (6, 8, 9). Our previous study established an interrelationship between ezrin and calpain I (10). Further support for a membrane-cytoskeleton linking role came from a study in which calpain I-mediated proteolysis liberated the apical localization of ezrin and prevented activation of acid secretion in the apical membrane without altering the gross cytology (10).
To delineate the membrane-cytoskeleton linking role of ezrin, several groups have tried to identify ERM-binding proteins using pull-down assays. These include identification of hyaluronate receptor CD44 (11), ICAM (intercellular adhesion molecule-1) (12), and EBP50 (13). Because PDZ (PSD-95/Discs Large-ZO-1) domains are known to mediate associations with integral membrane proteins, it was hypothesized that membrane attachment of ezrin is likely to be mediated via EBP50, a PDZ domain-containing protein.
A recent study has demonstrated important roles for PDZ domain-containing proteins during cell polarization (14). One of the major groups of PDZ proteins is the membrane-associated guanylate kinase proteins. These proteins usually contain a non-catalytic guanylate kinase domain in combination with PDZ and SH3 1 domains. Many also contain a 4.1B or hook domain that allows these membrane-associated guanylate kinase proteins to bind members of the band 4.1 superfamily, such as protein 4.1, and ERM proteins (15, 16). PALS1 (protein associated with Lin seven-1) (17), a membrane-associated guanylate kinase protein, binds to a PDZ domain protein of PATJ (PALS1-associated tight junction protein), a human homolog of Drosophila Discs Lost (DGL) (18). DGL is crucial for epithelial cell polarity and exists in complex with the apical polarity determinant Crumbs in flies. It remains to be established how the PALS1-PATJ-Crumbs protein complex interacts with the actin-based cytoskeleton at the apical membrane during polarity establishment and/or maintenance.
To delineate the structure-function relationship of ezrin in the activation of gastric acid secretion, we took advantage of our recent development of streptolysin O-permeabilized gastric glands and assessed the requirement of ezrin in parietal cell activation by addition of recombinant ezrin and its deletion mutants. Our study demonstrates that the integrity of ezrin is critical for parietal cell activation. To identify the apical localization signal, we expressed full-length ezrin and its deletion mutants tagged with green fluorescent protein (GFP) in the cultured parietal cells. Our studies indicate that the N terminus of ezrin (amino acids 1–50) is sufficient for its localization to the apical membrane, whereas overexpression of this 50-amino acid fragment liberates endogenous ezrin from the apical membrane. Our pull-down assay revealed an association between ezrin and PALS1. Elimination of PALS1 by small interfering RNA (siRNA) diminished the apical localization of ezrin and effected a dominant-negative role in parietal cell activation. We propose that ezrin-PALS1 interaction provides a link between the apical plasma membrane and actin-based cytoskeleton in parietal cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNA Construction—The bacterial expression vectors containing human ezrin fused to glutathione S-transferase (GST) were generous gifts from Dr. Monique Arpin. GFP-ezrin was constructed by ligating an EcoRI-SalI PCR-amplified ezrin cDNA into pEGFP-N1 (Clontech) as described previously (7). The ezrin deletion mutants were constructed by a standard PCR method using the following primers: ezrin-N1, 5'-TCG TCG ACG GTA CCG CGG GCC CGG GAT-3' (forward) and 5'-GAT GGT GTC AGG CTT CCT GCG GCG CAT-3') (reverse); ezrin-C1, 5'-ATG GAG GTG CAG CAG ATG AAG GCC CA-3 (forward) and 5'-CAG AAT TCG AAG CTT GAG CTC GAG A-3' (reverse); ezrin-N2, 5'-TCG TCG ACG GTA CCG CGG GCC CGG GAT-3' (forward) and 5'-CAC ATA GTG GAG GCC AAA GTA CCA-3' (reverse); and ezrin-C2, 5'-ATG ATC CAC AAC GAG AAC ATG A-3' (forward) and 5'-CAG AAT TCG AAG CTT GAG CTC GAG A-3' (reverse). All constructs were sequenced in full. GST-ezrin and its deletion mutation were constructed in pGEX-2T (Amersham Biosciences) as described (20).
Myelin basic protein (MBP)-tagged PALS1 was constructed in a modified pMAL-C2 vector as described by Fukata et al. (21). Briefly, PALS1 was amplified by PCR using primers 5'-CGG GAT CCA TGA CAA CAT CAT ATA TGA-3' and 5'-GGA ATT CTC ACC TTA GCC AGG TGG AT-3', digested with BamHI and EcoRI, and inserted into the pMAL-C2 vector digested with BamHI and EcoRI. Myc-tagged PALS1-(1–675), PALS1-(1–181), and PALS1-(182–675) constructs were generated as described previously (18).
Isolation of Gastric Glands and Aminopyrine Uptake Assay—Gastric glands were isolated from New Zealand White rabbits as described by Yao et al. (10). Briefly, rabbit stomach was perfused under high pressure with phosphate-buffered saline (PBS; 2.25 mM K2HPO4, 6 mM Na2HPO4, 1.75 mM NaH2PO4, and 136 mM NaCl) containing 1 mM CaCl2 and 1 mM MgSO4. The gastric mucosa was scraped from the smooth muscle layer, minced, and then washed twice with minimal essential medium (MEM) buffered with 20 mM HEPES (pH 7.4) (HEPES/MEM). The minced mucosa was digested with 15 mg of collagenase (Sigma). Intact gastric glands were collected from the digestion mixture for 20–25 min and then washed three times with HEPES/MEM. In all subsequent gland experiments (aminopyrine uptake assay), glands were resuspended at 5% (v/v) cytocrit in the appropriate buffer for final assay.
Stimulation of intact and streptolysin O (SLO)-permeabilized rabbit gastric glands was quantified using the aminopyrine uptake assay as described by Ammar et al. (22). Briefly, intact glands in HEPES/MEM were washed twice by settling at 4 °C in ice-cold K buffer (10 mM Tris base, 20 mM HEPES acid, 100 mM KCl, 20 mM NaCl, 1.2 mM MgSO4, 1 mM NaH2PO4, and 40 mM mannitol (pH 7.4)). SLO was added to a final concentration of 1 µg/ml, and the glands (at 5% cytocrit) were mixed by inversion and incubated on ice for 10 min. The glands were then washed twice with ice-cold K buffer to remove unbound SLO, and the permeabilization was initiated by incubating the gland suspension at 37 °C in K buffer containing 1 mM pyruvate and 10 mM succinate.
To evaluate the function of ezrin in parietal cell activation, we generated recombinant ezrin and its deletion mutants in bacteria. Briefly, GST-fused ezrin and deletion mutants were expressed in BL21(DE3) bacteria and purified using a glutathione affinity column as described (7). The fusion proteins were then eluted in PBS containing 10 mM glutathione and applied to a gel filtration column (Econ-Pac 10 DG, Bio-Rad) to remove glutathione and to exchange PBS for K buffer. The recombinant proteins were estimated to be 95% pure by SDS-PAGE; major contaminants were degraded fragments of ezrin. Protein concentrations were determined by the Bradford assay (23).
Affinity Precipitation of PALS1 and Ezrin—GST-ezrin-N2 was used as an affinity matrix to isolate proteins interacting with ezrin. A gastric membrane fraction from rabbit stomach was extracted with lysis buffer containing 50 mM Tris-HCl (pH 6.9), 100 mM NaCl, and 0.1% Triton X-100 plus proteinase inhibitor mixture (pepstatin A, leupeptin, aprotinin, and chymostatin at final concentrations of 5 µg/ml each). The cell lysates were clarified using an Eppendorf centrifuge at 13,000 rpm for 10 min. The resulting supernatant was incubated with 30 µl of GST-ezrin-N2 beads at room temperature for 2 h. The beads were then washed with lysis buffer three times, followed by boiling in 1x sample buffer. The samples were resolved on 6–16% gradient SDS-polyacrylamide gel and analyzed by Western blotting.
To characterize the interaction between PALS1 and ezrin, GST-tagged ezrin and deletion mutants C1 and N1 were used as an affinity matrix to isolate MBP-tagged recombinant PALS1 from bacteria cell lysates using the protocol described by Lou et al. (24). The beads were then washed with PBS three times, followed by boiling in 1x sample buffer. The samples were resolved on 6–16% gradient SDS-polyacrylamide gel and analyzed by Western blotting.
Cell Culture and Transfection—Primary cultures of gastric parietal cells from rabbit stomach were produced and maintained as described (7). Separate cultures of parietal cells were transfected with plasmids encoding GFP-tagged wild-type ezrin and/or deletion mutants using Lipofectamine 2000 according to the manufacturer's instructions. Briefly, 1 µg of DNA was incubated in 600 µl of Opti-MEM (antibiotic-free), and 6 µl of Lipofectamine 2000 was added and left at room temperature for 25 min. The cultured parietal cells (3% cytocrit; 6-well plates) were washed once with Opti-MEM. The DNA/lipid mixture was added to the plates and incubated for 4 h, followed by replacement with 1.5 ml of medium B (Dulbecco's modified Eagle's medium/F-12 (Invitrogen) supplemented with 20 mM HEPES, 0.2% bovine serum albumin, 10 mM glucose; 8 nM epidermal growth factor, 1x selenite, insulin, and transferrin (SITE) medium (Sigma), 1 mM glutamine, 100 units/ml penicillin-streptomycin, 400 µg/ml gentamicin sulfate, and 15 µg/liter geneticin or 20 µg/liter novobiocin, pH 7.4). The transfected cells were then maintained in culture at 37 °C until used for protein expression, partition, immunoprecipitation, or immunofluorescence.
To confirm the interaction between PALS1 and ezrin, GFP-ezrin- and FLAG-PALS1-cotransfected parietal cells were harvested and lysed in 1.5 ml of Tris-buffered saline (20 mM Tris-Cl, pH 7.4, 150 mM NaCl, 2 mM EGTA, and 0.1% Triton X-100) containing proteinase inhibitor mixture. The cell lysates were clarified using an Eppendorf centrifuge at 13,000 rpm for 10 min. The resulting supernatants were then incubated with 15 µg of anti-GFP monoclonal antibody JL-18 at room temperature for 2 h, followed by addition of 10 µl of protein A/G beads (Pierce) for an additional hour. The beads were collected and washed with Tris-buffered saline before boiling in SDS-PAGE sample buffer. Immunoprecipitates were then fractionated by SDS-PAGE, and proteins were transferred to nitrocellulose membrane for Western blot analyses. The blot was first labeled with anti-ezrin antibody 4A5 to verify the efficiency of GFP immunoprecipitation. The blot was then stripped with SDS-PAGE sample buffer at 55 °C for 20 min, followed by confirmation of PALS1 protein precipitated by GFP-ezrin using anti-FLAG monoclonal antibody M2.
siRNA Treatment and Assay for Knockdown Efficiency—The siRNA sequences used for silencing PALS1 correspond to coding regions 504–524 (siRNA-1) and 488–508 (siRNA-2). As a control, either a duplex targeting cyclophilin or a scrambled sequence was used (24). The 21-mer oligonucleotide RNA duplexes were synthesized by Dharmacon Research, Inc. (Boulder, CO). In trial experiments, different concentrations of siRNA oligonucleotides were used for different treatment times as detailed previously, and transfection efficiency was judged based on the uptake of fluorescein isothiocyanate-conjugated oligonucleotides (24). In brief, cultured parietal cells were transfected with siRNA-1 and siRNA-2 oligonucleotides or control scrambled oligonucleotides, and the efficiency of this siRNA-mediated protein suppression was judged by Western blot analysis.
Immunofluorescence Microscopy—For cytolocalization of exogenously expressed ezrin, cultured parietal cells were transfected with GFP, GFP-tagged wild-type ezrin, and GFP-tagged ezrin deletion mutants (N1, N2, C1, and C2) and maintained in MEM for 3036 h. Some cultures were treated with 100 µM cimetidine to maintain a resting state; others were treated with the secretory stimulants 100 µM histamine and 50 µM isobutylmethylxanthine in the presence of SCH28080 a proton pump inhibitor (7). Treated cells were then fixed with 2% formaldehyde for 10 min and washed three times with PBS, followed by permeabilization in 0.1% Triton X-100 for 5 min. Prior to application of the primary antibody, the fixed and permeabilized cells were blocked with 0.5% bovine serum albumin in PBS, followed by incubation with the primary antibody against ezrin (4A5) or GFP. The endogenous and exogenous ezrin proteins were labeled with fluorescein isothiocyanate-conjugated goat anti-mouse antibody and counterstained with rhodamine-coupled phalloidin to visualize filamentous actin. Coverslips were supported on slides by grease pencil markings and mounted in Vectashield (Vector Laboratories). Images were taken with a Zeiss Axiovert 200 fluorescence microscope using a 63 x 1.3 numerical aperture PlanApo objective. Figures were constructed using Adobe Photoshop.
Confocal Microscopy—Immunostained parietal cells were examined under a Zeiss LSM510 NLO laser scanning confocal microscope with the scan head mounted transversely on a Zeiss Axiovert 200 inverted microscope with a 40 x 1.0 numerical aperture PlanApo objective. Single images were collected by averaging 10 scans at a scan rate of 1 s/scan. Optical section series were collected with a spacing of 0.4 µm in the Z axis through an 12-µm thickness of the cultured parietal cells. The images from double labeling were simultaneously collected using a dichroic filter set with Zeiss LSM5 image processing software. Digital data were exported into Adobe Photoshop for presentation.
Western Blotting—Samples were subjected to SDS-PAGE on 616% gradient gel and transferred to nitrocellulose membrane. Proteins were probed with the appropriate primary antibodies and detected using an ECL kit (Pierce). The band intensity was then quantified using a PhosphorImager (Amersham Biosciences).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7% decrease), and there was no dose-dependent inhibitory effect. In contrast, N-terminal ezrin mutants caused a dose-dependent inhibition of acid secretion in SLO-permeabilized glands as measured by aminopyrine uptake. No significant inhibition was noted at 2.5 µg/ml protein, but 5 µg/ml protein caused a 23.9% reduction in acid secretion, and maximal inhibition (89–91%) occurred at 10 and 20 µg/ml (Fig. 1C). Interestingly, addition of C-terminal ezrin caused relatively small changes in aminopyrine uptake (at most, an 9.7% decrease), and there was no dose-dependent inhibitory effect. These experiments support the notion that the integrity of ezrin is required for parietal cell activation.
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Ezrin Interacts with PALS1 via Its N Terminus—Stimulation of parietal cells by histamine results in dramatic expansion of the apical canalicular plasma membrane due to insertion of H,K-ATPase-containing vesicular membranes. Our recent study demonstrated that ezrin couples activation of protein kinase A to the apical membrane-cytoskeleton remodeling associated with parietal cell activation (7). To identify the protein responsible for ezrin localization to the apical membrane, we employed GST-tagged recombinant ezrin as an affinity matrix to isolate proteins from the gastric epithelial cell lysates as described (13). To this end, GST-ezrin-N2 was immobilized on agarose beads, mixed with detergent-soluble tissue lysates, and washed extensively, and any binding proteins were eluted and fractionated on SDS-polyacrylamide gel. As shown in Fig. 3A (lane 3), there were two polypeptide bands with apparent molecular masses of 80–83 kDa that specifically bound to GST-ezrin-N2. Mass spectrometric analyses suggested that the 80-kDa band is ezrin, whereas the 83-kDa band is PALS1-like protein, a putative band 4.1-binding protein in the apical polarity determinant protein complex of flies (17). To confirm the results from the mass spectrometric analyses, we carried out Western blot analyses using ezrin- and PALS1-specific antibodies. As shown in Fig. 3B, immunoblot analyses confirmed that the 80-kDa band is ezrin, whereas the 83-kDa protein band is PALS1. This ezrin was probably recovered due to a direct association with immobilized ezrin-N2 or by virtue of being bound to PALS1. No actin was retained on the ezrin affinity beads. Thus, we concluded that PALS1 associates with either ezrin from gastric cells or recombinant ezrin-N2.
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To define the binding domain of ezrin responsible for its association with PALS1, we expressed and purified GST-tagged ezrin and ezrin deletion mutants C1 and N1 from bacteria using glutathione-agarose beads. Purified GST-ezrin proteins were used as an affinity matrix for absorbing MBP-tagged recombinant PALS1 protein from bacterial lysates. As shown in Fig. 3D, the association of recombinant PALS1 with ezrin was analyzed by immunoblotting using anti-PALS1 antibody. Recombinant PALS1 was absorbed by full-length ezrin and ezrin-N1 (Fig. 3D, lanes 2 and 4, respectively), but not by ezrin-C1, indicating that the N terminus of ezrin specifies the association between ezrin and PALS1. Therefore, we concluded that ezrin binds to PALS1 via its N terminus.
To identify the region of PALS1 that interacts with ezrin, we transfected parietal cells with Myc-tagged full-length N-terminal (amino acids 1–181) and C-terminal (amino acids 182–675) PALS1 and carried out Myc immunoprecipitation. As shown in Fig. 3E, full-length and C-terminal PALS1 pulled down ezrin from parietal cell lysates, which confirms the interaction between PALS1 and ezrin and offers preliminary mapping of the binding interfaces.
PALS1 Is Co-localized with Ezrin to the Apical Membrane in Parietal Cells—Because PALS1 is part of the apical polarity determinant protein complex conserved from flies to humans, the biochemical interaction between ezrin and PALS1 propelled us to test whether PALS1 is an apically distributed protein. Fig. 3F (panels a–c) shows optical sections from non-secreting (resting) parietal cells. Similar to what has been noted in earlier studies, ezrin was localized to the plasma membranes, most prominently to the apical membrane vacuoles sequestered to the cell interior as rings (Fig. 3F, panel b, red). PALS1 staining appeared as six clearly resolved rings of apical membrane vacuoles (Fig. 3F, panel a, green). The PALS1 signal was co-localized with ezrin as seen in the merged image from two channels (Fig. 3F, panel c, yellow). The co-distribution of PALS1 with ezrin to the apical membrane confirms the biochemical interaction between these two proteins.
The stimulation of parietal cell acid secretion involves insertion of H,K-ATPase-containing tubulovesicular membrane into apical canalicular membrane, resulting in dilation of apical membrane vacuoles as active HCl and water transport occur (7). Because of this swelling, stimulated parietal cells are considerably larger in diameter than their resting counterparts (7). Because PALS1 was co-distributed with ezrin to the apical membrane, we tested whether PALS1 and ezrin remain colocalized as the apical cytoskeleton remodeling occurs. Fig. 3F (panels d–f) shows optical sections taken from parietal cells treated with the secretagogues histamine and isobutylmethylxanthine and probed for PALS1 and ezrin. As seen in resting parietal cells (Fig. 3F, panels a–c), ezrin and PALS1 were primarily co-localized to the same regions in secreting cells. However, in the case of secreting cells, the dilated apical canalicular vacuoles occupied most of the cytoplasmic space (Fig. 3F, panels d and e), as noted in our recent study (7). Thus, PALS1 remains associated with ezrin at the apical plasma membrane of secreting parietal cells.
PALS1 Is Essential for the Localization of Ezrin to the Apical Membrane—Previous studies established the importance of the actin-based cytoskeleton (26) and the integrity of ezrin (10) in parietal cell activation. The function of ezrin in parietal cell activation has been directly demonstrated in our recent study (7). Given the observed interaction between PALS1 and ezrin in vitro and their co-distribution in vivo, it was of great interest to investigate the possible influence of PALS1 on the localization of ezrin to the apical membrane. To this end, we expressed Myc-tagged full-length and N-terminal PALS1 in gastric parietal cells and assessed their subcellular distribution profiles. Typically, we achieved a 3–4-fold expression in positively transfected parietal cells (7). As shown in Fig. 3G (panel a), Myc-tagged PALS1 was localized mainly to the parietal cell apical plasma membrane, which is superimposed with ezrin distribution (panel b). Despite an apical localization of N-terminal PALS1 (amino acids 1–181) (Fig. 3G, panel d), ezrin distribution became diffused in the PALS-(1–181)-transfected cells (panel e, green), suggesting that full-length PALS1 is required for ezrin localization to the apical membrane.
To test this hypothesis, we introduced siRNA oligonucleotides (siRNA-1) of PALS1 by transfection into parietal cells. To determine the efficient timing for knocking down PALS1 protein levels suppressed by the RNA interference, we transfected parietal cells with 100 nM siRNA-1 and collected cells at different post-transfection times. As shown in Fig. 4A (left panels), Western blotting with anti-PALS1 antibody revealed that siRNA-1 caused remarkable suppression of PALS1 protein levels at 48 h, whereas control cells treated with an irrelevant oligonucleotide (e.g. cyclophilin) expressed normal PALS1 levels. Quantitative analysis revealed that the siRNA-1 treatment caused a 5.9-fold suppression of PALS1 protein without altering the levels of other proteins such as actin. Further extension of the siRNA treatment interval to 72 h did not significantly improve the efficiency of PALS1 protein suppression. As PALS1 synthesis in 40% of the untransfected cells with little or no oligonucleotide was unlikely to be markedly diminished, the observed 5.9-fold inhibition at 100 nM must represent almost complete inhibition of PALS1 in 61 ± 3% of the successfully transfected cells. Another siRNA oligonucleotide (siRNA-2) targeted to a different sequence of PALS1 gave a similar suppression profile for PALS1 protein accumulation (Fig. 4A, right panels). This suppression was relatively specific, as it did not alter the levels of other proteins such as ezrin and actin.
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Treatment of parietal cells with siRNA-2 suppressed PALS1 protein accumulation and effected a dislocation of ezrin localization to the apical membrane (Fig. 4C, panel a') without alteration of F-actin and Cdc42 distribution (panels b' and d'). A control scrambled oligonucleotide did not alter the distribution profile of ezrin. Thus, we conclude that PALS1 is essential for the apical localization of ezrin, but not Cdc42, in gastric parietal cells.
Our previous study demonstrated that ezrin is essential for parietal cell activation (7). To confirm the function of ezrin-PALS1 association in the apical membrane, we examined the distribution of ezrin in histamine-stimulated parietal cells treated with a PALS1 siRNA oligonucleotide (siRNA-1). Fig. 4B shows optical sections taken from histamine-stimulated parietal cells simultaneously probed with fluorescein-conjugated anti-ezrin antibody 4A5 (panel e') and rhodamine-conjugated phalloidin (panel f'). In PALS1 siRNA-treated secreting cells, ezrin staining was distributed throughout the cytoplasm as noted in siRNA-treated resting cells, although we also noted some accumulation near the apical membrane vacuoles (Fig. 4B, panel g'). Experimentation using another siRNA oligonucleotide (siRNA-2) confirmed that PALS1 is essential for the apical localization of ezrin in parietal cells (Fig. 4C). Examination of F-actin staining revealed that the stimulation failed to dilate the apical canalicular vacuoles in the PALS1 siRNA-treated parietal cells (Fig. 4B, panel f'). However, in the case of the control siRNA-treated secreting cells, the dilated apical canalicular vacuoles occupied most of the cytoplasmic space (Fig. 4B, panels f and h). A previous study revealed that vacuole diameter can be used as a reporter for parietal cell secretory activity (34). Thus, we surveyed 83 cells from resting and stimulated populations transfected with either control or PALS1 siRNA (siRNA-1) oligonucleotides. The vacuolar measurements summarized in Table I show that stimulation dramatically extended vacuole diameter to 15.3 ± 1.3 µm in control oligonucleotide-treated parietal cells. However, the average vacuole diameter of PALS1-suppressed stimulated cells was only 4.7 ± 0.5 µm. We therefore conclude that ezrin-PALS1 interaction is essential for the dynamic remodeling of the apical cytoskeleton of parietal cells associated with stimulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A major role for the actin cytoskeleton in the secretory processes of parietal cells has been inferred from a study using actin disrupters that disorganize actin filaments and act to inhibit acid secretion (26). Highly organized microfilaments are typical features of microvilli at the apical membrane within the parietal cell canaliculus. In going from the resting to the secreting state, there are major changes at the apical canalicular surface, including elongation of microvilli. Interestingly, as the parietal cell returns to the resting state after withdrawal of stimulants, microfilament ultrastructural changes become apparent as a disorganization of actin filaments along with collapse of the apical canalicular surface (28, 29). These morphological studies indicate that reversible actin-based cytoskeletal dynamics are tightly linked to the secretory cycle in parietal cells. Our recent study demonstrated that ezrin couples protein kinase A-mediated phosphorylation to the remodeling of the apical membrane-cytoskeleton associated with acid secretion in parietal cells (7). However, our preliminary data showed that protein kinase A-mediated ezrin protein phosphorylation of Ser66 did not modulate ezrin-PALS1 interaction, consistent with our previous study in which alteration of Ser66 phosphorylation did not change ezrin distribution to the apical membrane (7). To search for ERM binding partners potentially involved in membrane association, Reczek et al. (13) use GST-ezrin as an affinity matrix to isolate a 50-kDa PDZ domain-containing phosphoprotein named EBP50 from human placental cell lysates. However, our search for EBP50 in isolated rabbit gastric parietal cells was unsuccessful (7). Thus, the interaction between ezrin and PALS1 established here represents a functional link between the ezrin molecule and the apical plasma membrane of gastric parietal cells. Our preliminary assessment of ezrin-PALS1 binding interface(s) revealed that the C-terminal region of PALS1 is essential for ezrin association. Precise mapping of the respective binding interfaces between these two proteins will help to delineate the molecular mechanisms underlying polarity establishment and/or maintenance in gastric parietal cells.
Parietal cell activation involves translocation of H,K-ATPase from the cytoplasm to the apical plasma membrane via multiple steps, including the possible trafficking over actin filaments, docking to secretory sites, insertion of the pump into the apical membrane, and perhaps maintenance of the pump in the apical membrane during active secretion. It will be essential to distinguish precisely where the ezrin-PALS1 interaction operates. Recent work has shown that the membrane fusion machinery component -SNAP (-soluble N-ethylmaleimide-sensitive factor attachment protein) is localized to the apical membrane of neuroepithelial cells (30). The -SNAP knockout experimentation effected a liberation of atypical protein kinase C, PALS1, and VAMP7 (vesicle-associated membrane protein-7) from the apical membrane, which suggests an interactive nature of the membrane fusion machinery and the apical membrane polarity determinants in neuroepithelial cells. Our previous study established the respective roles of the actin-based cytoskeleton and membrane fusion machinery in mediating H,K-ATPase translocation to the apical membrane (22); it remains to be established how the ezrin-PALS1 interaction operates the apical membrane-cytoskeleton dynamics to facilitate the docking and insertion of H,K-ATPase into the apical membrane of parietal cells for proton pumping.
In Drosophila, the Crumbs-Stardust-Discs Lost complex is required during the establishment of polarized epithelia. Embryos that lack a component of this complex or that overexpress Crumbs exhibit defects in epithelial morphogenesis (31–33). Two evolutionarily conserved multiprotein complexes, PALS1-PATJ-Crumbs and Cdc42-Par6-Par3-atypical protein kinase C, have been implicated in the polarization of Drosophila melanogaster epithelia. In fact, it has been shown that these two complexes are linked by Par6-PALS1 interaction (33). Our demonstration that suppression of PALS1 did not alter the apical distribution of Cdc42 and parietal cell polarity supports the notion that an alternative mechanism exists for linking Cdc42 to the apical plasma membrane (27). Interestingly, expression of C-terminal ezrin in cultured parietal cells or addition of C-terminal ezrin mutants to SLO-permeabilized glands did not effect a dominant action, suggesting that the recombinant proteins are unable to compete with endogenous protein for actin binding. In any event, further characterization of the ezrin-PALS1 apical membrane-cytoskeleton complex will provide detailed information regarding the molecular composition and structure-function relationships of the ezrin-containing apical signaling complex in cAMP-dependent polarized secretion in gastric parietal cells.
Taken together, the results presented in this work reveal that ezrin interacts with the evolutionarily conserved apical protein PALS1 and that this interaction specifies the apical localization of ezrin. Finally, we have shown that disruption of ezrin-PALS1 interaction blocks the remodeling of the apical membrane-cytoskeleton associated with the translocation and insertion of H,K-ATPase into the apical membrane. We propose that ezrin-PALS1 interaction links proton pump H,K-ATPase trafficking to the apical membrane-cytoskeleton remodeling required for polarized secretion in gastric parietal cells.
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FOOTNOTES |
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¶ Both authors contributed equally to this work.
A Georgia Cancer Coalition Distinguished Cancer Research Scholar. To whom correspondence should be addressed: School of Life Science, University of Science and Technology of China, Hefei 230027, People's Republic of China. E-mail: yaoxb{at}ustc.edu.cn
1 The abbreviations used are: SH3, Src homology 3; GFP, green fluorescent protein; siRNA, small interfering RNA; GST, glutathione S-transferase; MBP, myelin basic protein; PBS, phosphate-buffered saline; MEM, minimal essential medium; SLO, streptolysin O.
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ACKNOWLEDGMENTS |
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