FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2098570503> ?p ?o ?g. }

• W2098570503 endingPage "13592" @default.
• W2098570503 startingPage "13584" @default.
• W2098570503 abstract "The ERM (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 regulated HCl secretion in gastric parietal cells. Here, we show that the integrity of ezrin is essential for parietal cell activation and provide the first evidence that ezrin interacts with PALS1, an evolutionarily conserved PDZ and SH3 domain-containing protein. Our biochemical study verifies that ezrin binds to PALS1 via its N terminus and is co-localized with PALS1 to the apical membrane of gastric parietal cells. Furthermore, our study shows that PALS1 is essential for the apical localization of ezrin, as either suppression of PALS1 protein accumulation or deletion of the PALS1-binding domain of ezrin eliminated the apical localization of ezrin. Finally, our study demonstrates the essential role of ezrin-PALS1 interaction in the apical membrane remodeling associated with parietal cell secretion. Taken together, these results define a novel molecular mechanism linking ezrin to the conserved apical polarity complexes and their roles in polarized epithelial secretion of gastric parietal cells. The ERM (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 regulated HCl secretion in gastric parietal cells. Here, we show that the integrity of ezrin is essential for parietal cell activation and provide the first evidence that ezrin interacts with PALS1, an evolutionarily conserved PDZ and SH3 domain-containing protein. Our biochemical study verifies that ezrin binds to PALS1 via its N terminus and is co-localized with PALS1 to the apical membrane of gastric parietal cells. Furthermore, our study shows that PALS1 is essential for the apical localization of ezrin, as either suppression of PALS1 protein accumulation or deletion of the PALS1-binding domain of ezrin eliminated the apical localization of ezrin. Finally, our study demonstrates the essential role of ezrin-PALS1 interaction in the apical membrane remodeling associated with parietal cell secretion. Taken together, these results define a novel molecular mechanism linking ezrin to the conserved apical polarity complexes and their roles in polarized epithelial secretion of gastric parietal cells. The functions of an epithelium depend on the polarized organization of its individual epithelial cells. The acquisition of a fully polarized phenotype involves a cascade of complex events, including cell-cell adhesion, assembly of a lateral cortical complex, reorganization of the cytoskeleton, and polarized targeting of transport vesicles to the apical and basolateral membranes (1.Yeaman C. Grindstaff K.K. Hansen M.D. Nelson W.J. Curr. Biol. 1999; 9: R515-R517Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Ezrin is an actin-binding protein of the ERM (ezrin/radixin/moesin) family of membrane-cytoskeleton linker proteins (2.Bretscher A. Edwards K. Fehon R.G. Nat. Rev. Mol. Cell. Biol. 2002; 3: 586-599Crossref PubMed Scopus (1120) Google Scholar). Within the gastric epithelium, ezrin has been localized exclusively to parietal cells and primarily to the apical canalicular membrane of these cells (e.g. Refs. 3.Urushidani T. Hanzel D.K. Forte J.G. Am. J. Physiol. 1989; 256: G1070-G1081Crossref PubMed Google Scholar and 4.Yao X. Forte J.G. Annu. Rev. Physiol. 2003; 65: 103-131Crossref PubMed Scopus (213) Google Scholar). Our previous studies showed that gastric ezrin is co-distributed with the β-actin isoform in vivo (5.Yao X. Chaponnier C. Gabbiani G. Forte J.G. Mol. Biol. Cell. 1995; 6: 541-557Crossref PubMed Scopus (104) Google Scholar) and preferentially bound to the β-actin isoform in vitro (6.Yao X. Cheng L. Forte J.G. J. Biol. Chem. 1996; 271: 7224-7229Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). 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.Yao X. Forte J.G. Annu. Rev. Physiol. 2003; 65: 103-131Crossref PubMed Scopus (213) Google Scholar). Recently, we mapped the protein kinase A phosphorylation site on ezrin and illustrated the phosphoregulation of ezrin in gastric acid secretion (7.Zhou R. Cao X. Watson C. Miao Y. Guo Z. Forte J.G. Yao X. J. Biol. Chem. 2003; 278: 35651-35659Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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 α-helical and terminating in an ∼100-residue domain in which an F-actin-binding site resides (6.Yao X. Cheng L. Forte J.G. J. Biol. Chem. 1996; 271: 7224-7229Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 8.Turunen O. Wahlstrom T. Vaheri A. J. Cell Biol. 1994; 126: 1445-1453Crossref PubMed Scopus (343) Google Scholar, 9.Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (154) Google Scholar). Our previous study established an interrelationship between ezrin and calpain I (10.Yao X. Thibodeau A. Forte J.G. Am. J. Physiol. 1993; 265: C36-C46Crossref PubMed Google Scholar). 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.Yao X. Thibodeau A. Forte J.G. Am. J. Physiol. 1993; 265: C36-C46Crossref PubMed Google Scholar). 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.Tsukita S. Oishi K. Sato N. Sagara J. Kawai A. Tsukita S. J. Cell Biol. 1994; 126: 391-401Crossref PubMed Scopus (676) Google Scholar), ICAM (intercellular adhesion molecule-1) (12.Helander T.S. Carpen O. Turunen O. Kovanen P.E. Vaheri A. Timonen T. Nature. 1996; 382: 265-268Crossref PubMed Scopus (200) Google Scholar), and EBP50 (13.Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (512) Google Scholar). 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.Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1029) Google Scholar). 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 1The 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. 1The 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. 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.Lue R.A. Marfatia S.M. Branton D. Chishti A.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9818-9822Crossref PubMed Scopus (343) Google Scholar, 16.Cohen A.R. Woods D.F. Marfatia S.M. Walther Z. Chishti A.H. Anderson J.M. Wood D.F. J. Cell Biol. 1998; 142: 129-138Crossref PubMed Scopus (318) Google Scholar). PALS1 (protein associated with Lin seven-1) (17.Kamberov E. Makarova O. Roh M. Liu A. Karnak D. Straight S. Margolis B. J. Biol. Chem. 2000; 275: 11425-11431Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), 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.Roh M.H. Makarova O. Liu C.J. Shin K. Lee S. Laurinec S. Goyal M. Wiggins R. Margolis B. J. Cell Biol. 2002; 157: 161-172Crossref PubMed Scopus (291) Google Scholar). 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. Reagents—[14C]Aminopyrine was obtained from PerkinElmer Life Sciences. Monoclonal antibody JL-18 against GFP was purchased from Clontech (Palo Alto, CA), and anti-ezrin antibody 4A5 was produced as described by Hanzel et al. (19.Hanzel D.K. Urushidani T. Usinger W.R. Smolka A. Forte J.G. Am. J. Physiol. 1989; 256: G1082-G1089Crossref PubMed Google Scholar). Anti-FLAG monoclonal antibody M2 and rabbit anti-Cdc42 antibody were purchased from Sigma and Santa Cruz Biotechnology Inc. (Santa Cruz, CA), respectively. Rhodamine-coupled phalloidin and Alex Fluor 350-conjugated goat anti-rabbit IgG were purchased from Molecular Probes, Inc. (Eugene, OR). Lipofectamine 2000 was obtained from Invitrogen. 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.Zhou R. Cao X. Watson C. Miao Y. Guo Z. Forte J.G. Yao X. J. Biol. Chem. 2003; 278: 35651-35659Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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.Algrain M. Turunen O. Vaheri A. Louvard D. Arpin M. J. Cell Biol. 1993; 120: 129-139Crossref PubMed Scopus (372) Google Scholar). Myelin basic protein (MBP)-tagged PALS1 was constructed in a modified pMAL-C2 vector as described by Fukata et al. (21.Fukata M. Nakagawa M. Itoh N. Kawajiri A. Yamaga M. Kuroda S. Kaibuchi K. Mol. Cell. Biol. 2001; 21: 2165-2183Crossref PubMed Scopus (78) Google Scholar). 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.Roh M.H. Makarova O. Liu C.J. Shin K. Lee S. Laurinec S. Goyal M. Wiggins R. Margolis B. J. Cell Biol. 2002; 157: 161-172Crossref PubMed Scopus (291) Google Scholar). Isolation of Gastric Glands and Aminopyrine Uptake Assay—Gastric glands were isolated from New Zealand White rabbits as described by Yao et al. (10.Yao X. Thibodeau A. Forte J.G. Am. J. Physiol. 1993; 265: C36-C46Crossref PubMed Google Scholar). 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.Ammar D.A. Zhou R. Forte J.G. Yao X. Am. J. Physiol. 2002; 282: G23-G33Crossref PubMed Scopus (51) Google Scholar). 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.Zhou R. Cao X. Watson C. Miao Y. Guo Z. Forte J.G. Yao X. J. Biol. Chem. 2003; 278: 35651-35659Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). 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 1× 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.Lou Y. Yao J. Zereshki A. Dou Z. Ahmed K. Wang H. Hu J. Wang Y. Yao X. J. Biol. Chem. 2004; 279: 20049-20057Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The beads were then washed with PBS three times, followed by boiling in 1× 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.Zhou R. Cao X. Watson C. Miao Y. Guo Z. Forte J.G. Yao X. J. Biol. Chem. 2003; 278: 35651-35659Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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, 1× 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.Lou Y. Yao J. Zereshki A. Dou Z. Ahmed K. Wang H. Hu J. Wang Y. Yao X. J. Biol. Chem. 2004; 279: 20049-20057Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). 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.Lou Y. Yao J. Zereshki A. Dou Z. Ahmed K. Wang H. Hu J. Wang Y. Yao X. J. Biol. Chem. 2004; 279: 20049-20057Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). 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 30∼36 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.Zhou R. Cao X. Watson C. Miao Y. Guo Z. Forte J.G. Yao X. J. Biol. Chem. 2003; 278: 35651-35659Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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 × 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 × 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 6∼16% 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). Integrity of Ezrin Is Required for Parietal Cell Activation— The gastric parietal cell offers an excellent system to study ezrin function, when other ERM proteins are absent. We have recently established a permeable gland model that permits entry of relatively large molecular components (e.g. actin and syntaxin) (22.Ammar D.A. Zhou R. Forte J.G. Yao X. Am. J. Physiol. 2002; 282: G23-G33Crossref PubMed Scopus (51) Google Scholar). Our previous study suggested that the integrity of ezrin is required for parietal cell activation, as calpain-mediated proteolysis of ezrin is correlated with inhibition of acid secretion (10.Yao X. Thibodeau A. Forte J.G. Am. J. Physiol. 1993; 265: C36-C46Crossref PubMed Google Scholar). To directly assess the role of ezrin in parietal cell activation, we introduced recombinant full-length ezrin and its deletion mutants into SLO-permeabilized glands. Recombinant proteins expressed in bacteria were purified to homogeneity using a glutathione column (Fig. 1B). Recombinant proteins were added to the SLO-permeabilized glands in the presence and absence of cAMP/ATP. Addition of full-length ezrin caused relatively small changes in aminopyrine uptake (at most, an ∼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. The N Terminus of Ezrin Specifies Its Apical Localization in Parietal Cells—We reasoned that the inhibition seen in the SLO-permeabilized glands is due to a dominant effect exerted by addition of ezrin deletion mutants. Because ezrin is postulated to be a membrane-cytoskeleton linker, we next searched for the apical targeting domain of ezrin. Our previous study showed that exogenously expressed ezrin bears the same biochemical characteristics as those of endogenous protein (7.Zhou R. Cao X. Watson C. Miao Y. Guo Z. Forte J.G. Yao X. J. Biol. Chem. 2003; 278: 35651-35659Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). To determine whether there were any major changes in the behavior of exogenously expressed GFP-ezrin mutants, we measured the partitioning of exogenously expressed full-length ezrin and its deletion mutants into the digitonin-soluble fraction compared with the insoluble “cytoskeletal” fraction based on the Western blot analyses using anti-GFP antibody. As summarized in Fig. 2A, 63.3 ± 4.5% of full-length ezrin resided in the digitonin-insoluble fraction, consistent with previous reports (e.g. Refs. 7.Zhou R. Cao X. Watson C. Miao Y. Guo Z. Forte J.G. Yao X. J. Biol. Chem. 2003; 278: 35651-35659Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar and 25.Hanzel D. Reggio H. Bretscher A. Forte J.G. Mangeat P. EMBO J. 1991; 10: 2363-2373Crossref PubMed Scopus (151) Google Scholar). Partitioning of GFP-ezrin-N1 and GFP-ezrin-N2 was similar to that of full-length ezrin; 62.1 ± 4.7% of ezrin-N1 and 60.7 ± 5.1% of ezrin-N2 were associated with the digitonin-insoluble fraction. Interestingly, the majority of GFP-ezrin-C1 and GFP-ezrin-C2 was partitioned into soluble fractions: 87.5 ± 7.3% of ezrin-C1 and 89.7 ± 8.7% of ezrin-C2 were distributed to the digitonin-soluble fraction. The distinct distribution profiles between the N- and C-terminal deletion mutants may reflect their difference in modulating acid secretion in SLO-permeabilized glands. To determine the subcellular localization of ezrin deletion mutants in parietal cells, the transfected cells were double-stained for GFP using anti-GFP monoclonal antibody (green) and for F-actin using phalloidin (red). Fig. 2B shows optical sections from GFP-ezrin-transfected cells, all maintained in the non-secreting state. Similar to what has been noted in a recent study (7.Zhou R. Cao X. Watson C. Miao Y. Guo Z. Forte J.G. Yao X. J. Biol. Chem. 2003; 278: 35651-35659Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), full-length exogenous GFP-ezrin was localized to the plasma membranes, most prominently to the apical membrane vacuoles sequestered to the cell interior and somewhat more sparsely to the basolateral membrane surrounding the cells. The GFP-ezrin signal was relatively co-localized with F-actin (Fig. 2B, panels a–c). The distribution of the signal for the GFP-tagged N-terminal ezrin mutant constructs (N1 and N2) was similar to that for full-length ezrin, i.e. primarily associated with apical membrane vacuoles and to a lesser extent with the basolateral membrane (Fig. 2B, panels d and g). The distribution of F-actin also was not altered by the transfections (Fig. 2B, panels b, e, and h). However, the distribution of the signal for the GFP-tagged C-terminal ezrin mutants (C1 and C2) was distinctly different from that for full-length ezrin, i.e. primarily diffused throughout the cytoplasm, with no apparent localization to the apical membrane (Fig. 2B, panels j and m). Whereas the distribution of F-actin was also not altered by the transfections (Fig. 2B, panels k and n), the distribution of ezrin-C1 and ezrin-C2 no longer co-localized with actin (Fig. 2B, panels l and o). These data demonstrate that transfected GFP-tagged ezrin and N-terminal ezrin fragments are sufficient to localize to the apical plasma membrane of parietal cells. 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.Zhou R. Cao X. Watson C. Miao Y. Guo Z. Forte J.G. Yao X. J. Biol. Chem. 2003;" @default.
• W2098570503 created "2016-06-24" @default.
• W2098570503 creator A5001228727 @default.
• W2098570503 creator A5025578720 @default.
• W2098570503 creator A5033096483 @default.
• W2098570503 creator A5035642456 @default.
• W2098570503 creator A5040923571 @default.
• W2098570503 creator A5043338234 @default.
• W2098570503 creator A5056755738 @default.
• W2098570503 creator A5058780708 @default.
• W2098570503 creator A5058882529 @default.
• W2098570503 creator A5062720676 @default.
• W2098570503 creator A5063160228 @default.
• W2098570503 creator A5067643729 @default.
• W2098570503 creator A5077871658 @default.
• W2098570503 date "2005-04-01" @default.
• W2098570503 modified "2023-10-13" @default.
• W2098570503 title "PALS1 Specifies the Localization of Ezrin to the Apical Membrane of Gastric Parietal Cells" @default.
• W2098570503 cites W1427356251 @default.
• W2098570503 cites W1975862854 @default.
• W2098570503 cites W1983158430 @default.
• W2098570503 cites W1989170134 @default.
• W2098570503 cites W1993941425 @default.
• W2098570503 cites W1994095931 @default.
• W2098570503 cites W1995811820 @default.
• W2098570503 cites W1996275548 @default.
• W2098570503 cites W2005252392 @default.
• W2098570503 cites W2009671571 @default.
• W2098570503 cites W2024028178 @default.
• W2098570503 cites W2050482803 @default.
• W2098570503 cites W2060061784 @default.
• W2098570503 cites W2066562134 @default.
• W2098570503 cites W2067241536 @default.
• W2098570503 cites W2077159019 @default.
• W2098570503 cites W2083609148 @default.
• W2098570503 cites W2090424607 @default.
• W2098570503 cites W2105006060 @default.
• W2098570503 cites W2114964393 @default.
• W2098570503 cites W2117822148 @default.
• W2098570503 cites W2119785069 @default.
• W2098570503 cites W2125305131 @default.
• W2098570503 cites W2133862855 @default.
• W2098570503 cites W2139960392 @default.
• W2098570503 cites W2164890520 @default.
• W2098570503 cites W2165324125 @default.
• W2098570503 cites W2199402133 @default.
• W2098570503 cites W2258337558 @default.
• W2098570503 cites W2396170225 @default.
• W2098570503 cites W2432513752 @default.
• W2098570503 cites W311461775 @default.
• W2098570503 cites W4293247451 @default.
• W2098570503 doi "https://doi.org/10.1074/jbc.m411941200" @default.
• W2098570503 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15677456" @default.
• W2098570503 hasPublicationYear "2005" @default.
• W2098570503 type Work @default.
• W2098570503 sameAs 2098570503 @default.
• W2098570503 citedByCount "40" @default.
• W2098570503 countsByYear W20985705032012 @default.
• W2098570503 countsByYear W20985705032013 @default.
• W2098570503 countsByYear W20985705032014 @default.
• W2098570503 countsByYear W20985705032015 @default.
• W2098570503 countsByYear W20985705032016 @default.
• W2098570503 countsByYear W20985705032017 @default.
• W2098570503 countsByYear W20985705032019 @default.
• W2098570503 countsByYear W20985705032020 @default.
• W2098570503 countsByYear W20985705032021 @default.
• W2098570503 crossrefType "journal-article" @default.
• W2098570503 hasAuthorship W2098570503A5001228727 @default.
• W2098570503 hasAuthorship W2098570503A5025578720 @default.
• W2098570503 hasAuthorship W2098570503A5033096483 @default.
• W2098570503 hasAuthorship W2098570503A5035642456 @default.
• W2098570503 hasAuthorship W2098570503A5040923571 @default.
• W2098570503 hasAuthorship W2098570503A5043338234 @default.
• W2098570503 hasAuthorship W2098570503A5056755738 @default.
• W2098570503 hasAuthorship W2098570503A5058780708 @default.
• W2098570503 hasAuthorship W2098570503A5058882529 @default.
• W2098570503 hasAuthorship W2098570503A5062720676 @default.
• W2098570503 hasAuthorship W2098570503A5063160228 @default.
• W2098570503 hasAuthorship W2098570503A5067643729 @default.
• W2098570503 hasAuthorship W2098570503A5077871658 @default.
• W2098570503 hasBestOaLocation W20985705031 @default.
• W2098570503 hasConcept C105702510 @default.
• W2098570503 hasConcept C142669718 @default.
• W2098570503 hasConcept C1491633281 @default.
• W2098570503 hasConcept C185592680 @default.
• W2098570503 hasConcept C2779422922 @default.
• W2098570503 hasConcept C2779743957 @default.
• W2098570503 hasConcept C2780246058 @default.
• W2098570503 hasConcept C2781443798 @default.
• W2098570503 hasConcept C3018436504 @default.
• W2098570503 hasConcept C41625074 @default.
• W2098570503 hasConcept C55493867 @default.
• W2098570503 hasConcept C86803240 @default.
• W2098570503 hasConcept C95444343 @default.
• W2098570503 hasConceptScore W2098570503C105702510 @default.
• W2098570503 hasConceptScore W2098570503C142669718 @default.
• W2098570503 hasConceptScore W2098570503C1491633281 @default.
• W2098570503 hasConceptScore W2098570503C185592680 @default.

• next