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• W2038962261 abstract "ADP-ribosylation factor-related protein 1 (ARFRP1) plays a specific role in Golgi function controlling recruitment of GRIP domain proteins and ARL1 to the trans-Golgi. Deletion of the mouse Arfrp1 gene causes embryonic lethality during early gastrulation, because epiblast cells detach from the ectodermal cell layer and do not differentiate to mesodermal tissue. Here we show that in Arfrp1-/- embryos E-cadherin is mistargeted to intracellular compartments, whereas in control embryos it is present at the cell surface of trophectodermal and ectodermal cells. In enterocytes of intestine-specific Arfrp1 null mutants (Arfrp1vil-/-), E-cadherin is associated with intracellular membranes, partially colocalizing with the cis-Golgi marker GM130 or with punctae close to the cell surface. In contrast, in control enterocytes E-cadherin is exclusively located in the lateral membranes. In addition, ARL1 is dislocated from Golgi membranes to the cytosol of Arfrp1vil-/- enterocytes. Depletion of endogenous ARFRP1 by RNA interference leads to a dislocation of E-cadherin from the cell surface in HeLa cells and to a reduced cell aggregation in Ltk-Ecad cells. ARFRP1 was coimmunoprecipitated in a complex with E-cadherin, α-catenin, β-catenin, γ-catenin, and p120ctn from lysates of Madin-Darby canine kidney cells stably expressing myc-ARFRP1. These data indicate that knock-out of Arfrp1 disrupts the trafficking of E-cadherin through the Golgi and suggest an essential role of the GTPase in trans-Golgi network function. ADP-ribosylation factor-related protein 1 (ARFRP1) plays a specific role in Golgi function controlling recruitment of GRIP domain proteins and ARL1 to the trans-Golgi. Deletion of the mouse Arfrp1 gene causes embryonic lethality during early gastrulation, because epiblast cells detach from the ectodermal cell layer and do not differentiate to mesodermal tissue. Here we show that in Arfrp1-/- embryos E-cadherin is mistargeted to intracellular compartments, whereas in control embryos it is present at the cell surface of trophectodermal and ectodermal cells. In enterocytes of intestine-specific Arfrp1 null mutants (Arfrp1vil-/-), E-cadherin is associated with intracellular membranes, partially colocalizing with the cis-Golgi marker GM130 or with punctae close to the cell surface. In contrast, in control enterocytes E-cadherin is exclusively located in the lateral membranes. In addition, ARL1 is dislocated from Golgi membranes to the cytosol of Arfrp1vil-/- enterocytes. Depletion of endogenous ARFRP1 by RNA interference leads to a dislocation of E-cadherin from the cell surface in HeLa cells and to a reduced cell aggregation in Ltk-Ecad cells. ARFRP1 was coimmunoprecipitated in a complex with E-cadherin, α-catenin, β-catenin, γ-catenin, and p120ctn from lysates of Madin-Darby canine kidney cells stably expressing myc-ARFRP1. These data indicate that knock-out of Arfrp1 disrupts the trafficking of E-cadherin through the Golgi and suggest an essential role of the GTPase in trans-Golgi network function. GTPases of the ADP-ribosylation factor (ARF) 3The abbreviations used are: ARF, ADP-ribosylation factor; TGN, trans-Golgi network; MDCK, Madin-Darby canine kidney cells; ARL, ARF-like protein; RT, reverse transcription; qRT, quantitative RT; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; siRNA, small interfering RNA; shRNA, short hairpin RNA; ED, embryonic day; ES, embryonic stem cell. 3The abbreviations used are: ARF, ADP-ribosylation factor; TGN, trans-Golgi network; MDCK, Madin-Darby canine kidney cells; ARL, ARF-like protein; RT, reverse transcription; qRT, quantitative RT; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; siRNA, small interfering RNA; shRNA, short hairpin RNA; ED, embryonic day; ES, embryonic stem cell. family operate as molecular switches in the regulation of vesicular trafficking and organelle structure (1.Kahn R.A. Ridley A. Proteins and Cell Regulation. Kluwer Academic Publishers, Norwell, MA2003Google Scholar, 2.Kahn R.A. Cherfils J. Elias M. Lovering R.C. Munro S. Schürmann A. J. Cell Biol. 2006; 172: 645-650Crossref PubMed Scopus (197) Google Scholar). The ARF family includes three different groups of proteins, the ARFs, the ARLs (ARF-like proteins), and the secretion-associated Ras-related proteins. ARF-related protein 1 (ARFRP1) is a 25-kDa GTPase and member of the ARL family (2.Kahn R.A. Cherfils J. Elias M. Lovering R.C. Munro S. Schürmann A. J. Cell Biol. 2006; 172: 645-650Crossref PubMed Scopus (197) Google Scholar, 3.Schürmann A. Massmann S. Joost H.-G. J. Biol. Chem. 1995; 270: 30657-30663Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In contrast to other ARFs and ARLs, ARFRP1 can hydrolyze GTP in the absence of a GTPase-activating protein and lacks the N-myristoylation site (glycine 2), which is required for membrane association (3.Schürmann A. Massmann S. Joost H.-G. J. Biol. Chem. 1995; 270: 30657-30663Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). For the closest relative of ARFRP1, the yeast Arl3p protein, it was shown recently that membrane association is mediated via acetylation of the N-terminal methionine residue (4.Behnia R. Panic B. Whyte J.R. Munro S. Nat. Cell Biol. 2004; 6: 405-413Crossref PubMed Scopus (179) Google Scholar, 5.Setty S.R. Strochlic T.I. Tong A.H. Boone C. Burd C.G. Nat. Cell Biol. 2004; 6: 414-419Crossref PubMed Scopus (136) Google Scholar). ARFRP1 interacts with the Sec7 domain of the ARF-specific guanine nucleotide exchange factor cytohesin 1 in a GTP-dependent manner. This interaction resulted in the inhibition of the ARF/Sec7-dependent activation of phospholipase D in vitro and in vivo (6.Schürmann A. Schmidt M. Asmus M. Bayer S. Fliegert F. Koling S. Massmann S. Schilf C. Subauste M.C. Voss M. Jakobs K.H. Joost H.-G. J. Biol. Chem. 1999; 274: 9744-9751Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar).We and others have recently shown that ARFRP1 as well as its yeast ortholog Arl3p specifically control targeting of ARL1 and its effector Golgin-245 to the trans-Golgi (7.Setty S.R. Shin M.E. Yoshino A. Marks M.S. Burd C.G. Curr. Biol. 2003; 13: 401-404Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 8.Panic B. Whyte J.R. Munro S. Curr. Biol. 2003; 13: 405-410Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 9.Shin H.W. Kobayashi H. Kitamura M. Waguri S. Suganuma T. Uchiyama Y. Nakayama K. J. Cell Sci. 2005; 118: 4039-4048Crossref PubMed Scopus (56) Google Scholar, 10.Zahn C. Hommel A. Lu L. Hong W. Walther D.J. Florian S. Joost H.-G. Schürmann A. Mol. Membr. Biol. 2006; 23: 475-485Crossref PubMed Scopus (39) Google Scholar). GTP-bound ARFRP1 (ARFRP1-Q79L mutant) was associated with Golgi membranes and colocalized with ARL1. In contrast, the guanine nucleotide exchange defective ARFRP1 mutant (ARFRP1-T31N) clustered within the cytosol. Expression of ARFRP1-T31N or depletion of endogenous ARFRP1 by RNA interference disrupted the Golgi association of ARL1 and the GRIP domain protein Golgin-245 and altered the distribution of a trans-Golgi network (TGN) marker, syntaxin 6 indicating that ARFRP1 plays an important role for TGN structure and function (10.Zahn C. Hommel A. Lu L. Hong W. Walther D.J. Florian S. Joost H.-G. Schürmann A. Mol. Membr. Biol. 2006; 23: 475-485Crossref PubMed Scopus (39) Google Scholar).Deletion of Arfrp1 in mice resulted in embryonic lethality (11.Mueller A.G. Moser M. Kluge R. Leder S. Blum M. Büttner R. Joost H.-G. Schürmann A. Mol. Cell. Biol. 2002; 22: 1488-1494Crossref PubMed Scopus (33) Google Scholar). Arfrp1-/- blastocysts implanted in vivo and formed egg cylinder-stage embryos that appeared normal until day 5. During early gastrulation (at day 6–6.5), Arfrp1-/- embryos exhibited profound alterations of the distal part of the egg cylinder. Rounded pyknotic cells within this area were only loosely attached to the ectodermal cell layer, and some apoptotic cells were found in the proamniotic cavity. This observation suggested that ARFRP1 plays a critical role in processes during early gastrulation such as adhesion-dependent morphogenesis, cytoskeletal reorganization, and/or development of cell polarity (11.Mueller A.G. Moser M. Kluge R. Leder S. Blum M. Büttner R. Joost H.-G. Schürmann A. Mol. Cell. Biol. 2002; 22: 1488-1494Crossref PubMed Scopus (33) Google Scholar).Specific contacts of cells to the extracellular matrix and to neighboring cells are fundamental for embryogenesis, survival, and wound repair. Cadherins represent a large family of cell-cell adhesion proteins that play crucial roles in tissue patterning, cellular growth control, and in the regulation of cell shape and migration (12.Vleminckx K. Kemler R. BioEssays. 1999; 21: 211-220Crossref PubMed Scopus (302) Google Scholar, 13.Angst B.D. Marcozzi C. Magee A.I. J. Cell Sci. 2001; 114: 625-626Crossref PubMed Google Scholar, 14.Perez-Moreno M. Jamora C. Fuchs E. Cell. 2003; 112: 535-548Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar). Changes in cadherin expression are associated with numerous developmental events such as epithelial-mesenchymal transitions, e.g. during gastrulation each member of the family exhibits a specific spatial and temporal expression pattern (15.Hatta K. Takagi S. Fujisawa H. Takeichi M. Dev. Biol. 1987; 120: 215-227Crossref PubMed Scopus (434) Google Scholar). E-cadherin, the prototypical member of the classic cadherin family, is a major component of epithelial adherens junctions, where it mediates cell-cell adhesion through calcium-dependent, homophilic binding between molecules on adjacent cells (13.Angst B.D. Marcozzi C. Magee A.I. J. Cell Sci. 2001; 114: 625-626Crossref PubMed Google Scholar, 16.Leckband D. Structure (Camb.). 2002; 10: 739-740Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 17.Wheelock M.J. Johnson K.R. Annu. Rev. Cell Dev. Biol. 2003; 19: 207-235Crossref PubMed Scopus (524) Google Scholar). At the adherens junction, E-cadherin is bound to catenins with β-catenin attached to the cytoplasmic domain of E-cadherin and α-catenin associated with β-catenin. In contrast to previous models, α-catenin does not directly link the cadherin-catenin complex to the actin cytoskeleton (18.Drees F. Pokutta S. Yamada S. Nelson W.J. Weis W.I. Cell. 2005; 123: 903-915Abstract Full Text Full Text PDF PubMed Scopus (771) Google Scholar, 19.Yamada S. Pokutta S. Drees F. Weis W.I. Nelson W.J. Cell. 2005; 123: 889-901Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar). Recently, EPLIN/Lima-1 was identified as a missing link between the cadherin-catenin complex and the actin cytoskeleton (20.Abe K. Takeichi M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 13-19Crossref PubMed Scopus (277) Google Scholar). p120ctn binds to a juxtamembrane site in the cytoplasmic tail of E-cadherin (21.Yap A.S. Niessen C.M. Gumbiner B.M. J. Cell Biol. 1998; 141: 779-789Crossref PubMed Scopus (463) Google Scholar), and several roles of p120ctn modulating cadherin function have been discussed in the literature (22.Bryant D.M. Stow J.L. Trends Cell Biol. 2004; 14: 427-434Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). p120ctn is implicated to be involved in exocytosis, endocytosis, and turnover of cadherins (23.Chen X. Kojima S. Borisy G.G. Green K.J. J. Cell Biol. 2003; 163: 547-557Crossref PubMed Scopus (209) Google Scholar, 24.Davis M.A. Ireton R.C. Reynolds A.B. J. Cell Biol. 2003; 163: 525-534Crossref PubMed Scopus (567) Google Scholar, 25.Xiao K. Allison D.F. Buckley K.M. Kottke M.D. Vincent P.A. Faundez V. Kowalczyk A.P. J. Cell Biol. 2003; 163: 535-545Crossref PubMed Scopus (344) Google Scholar, 26.Xiao K. Garner J. Buckley K.M. Vincent P.A. Chiasson C.M. Dejana E. Faundez V. Kowalczyk A.P. Mol. Biol. Cell. 2005; 16: 5141-5151Crossref PubMed Scopus (215) Google Scholar).In this study, we tested the hypothesis that ARFRP1 modulates cadherin-mediated adhesion processes. We find ARFRP1 in a complex with E-cadherin, β-catenin, α-catenin, γ-catenin, and p120ctn. Additional data suggest that ARFRP1 is required for cell surface localization of E-cadherin because in the absence of ARFRP1, E-cadherin is dislocated from the plasma membrane, and cell adhesion is markedly reduced in vivo and in vitro.EXPERIMENTAL PROCEDURESAntisera—The polyclonal antibody against recombinant GST-ARFRP1 was described before (3.Schürmann A. Massmann S. Joost H.-G. J. Biol. Chem. 1995; 270: 30657-30663Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and used for immunohistochemistry (10.Zahn C. Hommel A. Lu L. Hong W. Walther D.J. Florian S. Joost H.-G. Schürmann A. Mol. Membr. Biol. 2006; 23: 475-485Crossref PubMed Scopus (39) Google Scholar). Affinity-purified polyclonal antiserum against recombinant ARL1 (E6P3; 0.3 μg/μl) was described before (27.Lowe S.L. Wong S.H. Hong W. J. Cell Sci. 1996; 10: 209-220Google Scholar, 28.Lu L. Hong W. Mol. Biol. Cell. 2003; 14: 3767-3781Crossref PubMed Scopus (122) Google Scholar) and used in a dilution of 1:100. The anti-GLUT1 antisera were raised against the C terminus of rat-GLUT1, as described previously (29.Weiland M. Schürmann A. Schmidt W.E. Joost H.G. Biochem. J. 1990; 270: 331-336Crossref PubMed Scopus (71) Google Scholar), and used in a dilution of 1:8000. The anti-GLUT2 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used in a dilution of 1:20; anti-DDP4 (ab28341) was purchased from Abcam (Cambridge, UK) and used in a dilution of 1:50. Anti-gp84 antibody against E-cadherin (30.Kasper M. Huber O. Grossmann H. Rudolph B. Tränkner C. Müller M. Histochem. Cell Biol. 1995; 104: 383-390Crossref PubMed Scopus (35) Google Scholar) was used in a dilution of 1:1000. Monoclonal antibody against E-cadherin (clone HECD-1) was used in a dilution of 1:400 for immunofluorescence analysis (Zymed Laboratories Inc.). The antibody against α-catenin was purchased from NanoTools (Teningen, Germany). The anti-Na+/K+-ATPase antibody (1:200) and the 58K Golgi marker (1:200) were purchased from Abcam (Cambridge, UK). Antibodies against p120ctn (1 μg/ml for Western blotting), IQGAP1 (1 μg/ml for Western blotting), and β-catenin (1:2500 for Western blotting) were purchased from BD Transduction Laboratories. Alexa Fluor® 546 F(ab′)2 fragment of goat anti-mouse IgG (H+L) or Alexa Fluor® 488 F(ab′)2 fragment of goat anti-rabbit IgG (H+L) (Molecular Probes, Eugene, OR) was used in a dilution of 1:800 as secondary antibodies.Cell Culture and Transient Transfection—HeLa cells were cultured in minimum essential medium with Earle's salts plus 10% (v/v) FCS. For aggregation assays, Ltk-Ecad cells were grown in Dulbecco's modified Eagle's medium (DMEM) high glucose in the presence of 10% (v/v) FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin at 5% CO2. Transient transfections of cells were performed with Lipofectamine 2000 (Invitrogen) according to manufacturer's protocols.MDCK T23 Cells and Stable Transfection of Myc-Arfrp1— The MDCK T23 cell line, which stably expresses the tetracycline-repressible transactivator, was described earlier (31.Barth A.I. Pollack A.L. Altschuler Y. Mostov K.E. Nelson W.J. J. Cell Biol. 1997; 136: 693-706Crossref PubMed Scopus (206) Google Scholar) and was kindly provided by Prof. Keith E. Mostov (Department of Anatomy, University of California, San Francisco). MDCK T23 cells were maintained in DMEM with 10% FCS supplemented with the necessary antibiotics and cultured under continuous presence of 40 ng/ml doxycycline. The medium was renewed every 48 h. N-terminal Myc tag was fused to the mouse Arfrp1 open reading frame by PCR and cloned into the pTRE2hyg vector (Clontech). Transfection of MDCK T23 cells was performed in 6-well plates with Lipofectamine 2000 according to the manufacturer's instructions. Twenty four hours after transfection, cells were reseeded into 10-cm dishes, and selection of transfected cells was achieved with 40 ng/ml doxycycline and 300 μg/ml hygromycin in DMEM. After selection for 12 days, surviving colonies were isolated with the use of cloning rings and expanded in 48 wells. At confluency, cells from each of the surviving clones were split and maintained in the presence or absence of doxycycline. ARFRP1 expression was assessed by immunofluorescence microscopy 48 h after removal of doxycycline. Clones positive for ARFRP1 expression were expanded, and inducible expression was confirmed by Western blot analysis.Immunocytochemistry and Indirect Immunofluorescence Microscopy—At the indicated time points, cells were washed with PBS and fixed with methanol (-20 °C for 10 min). Cells were washed with PBS, blocked with PBS, 0.1% (v/v) Tween 20:5% (v/v) normal goat serum for 20 min at room temperature and incubated with primary antibodies in antibody diluent (Dako, Glostrup, Denmark) for 1 h at room temperature. After extensive washing with PBS, 0.1% (v/v) Tween 20, cells were incubated with Alexa Fluor® 488- or Alexa Fluor® 546-conjugated secondary antibodies in antibody diluent at room temperature for 30 min. After washing with PBS, 0.1% (v/v) Tween 20, cells were mounted in fluorescent mounting medium (Dako) and analyzed with a Leica TCS SP2 Laser Scan inverted microscope. We scanned the cells sequentially with an argon-krypton laser (488 nm) to excite the Alexa 488 dye, and with a helium-neon laser (543 nm) to excite the Alexa 546 dye. The spectral detector recorded light emission at 510–560 and 580–660 nm, respectively. We processed images of 1024 × 1024 pixels with Adobe Photoshop CS (version 8.0.1).Generation of Intestine-specific Arfrp1 Null Mutants (Arfrp1vil-/-Mice)—For tissue-specific disruption of Arfrp1, we used the Cre/loxP recombination system and generated Arfrp1flox/flox mice in which exons 2 and 4 of Arfrp1 were flanked with loxP sites. The targeting vector also contained pGKneo/HSVtk cassette (Neo/tk) with a third loxP site that was introduced between exon 4 and 5. It was electroporated into embryonic stem (ES) cells that were screened for homologous recombination. A homologous recombined ES cell clone containing the targeted allele was retransfected with pIC-Cre to generate ES cell clones carrying the floxed Arfrp1 allele. One ES cell clone was injected into blastocysts, which were subsequently transferred into a day 2.5 pseudopregnant female C57BL/6 mouse. Male chimeric mice were mated with C57BL/6 females to generate Arfrp1flox/+ mice. Arfrp1flox/+ mice were backcrossed with C57BL/6 three times. Intestine-specific Arfrp1 null mutants (Arfrp1vil-/-) were generated by intercrossing Arfrp1flox/flox with transgenic mice that express Cre recombinase under the control of the villin promotor/enhancer (villin-Cre) (32.Madison B.B. Dunbar L. Qiao X.T. Braunstein K. Braunstein E. Gumucio D.L. J. Biol. Chem. 2002; 277: 33275-33283Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar). The animals were housed in a controlled environment (20 ± 2 °C, 12:12-h light/dark cycle) and had free access to water and standard chow diet. All animal experiments were approved by the Ethics Committee of the Ministry of Agriculture, Nutrition, and Forestry (State of Brandenburg, Germany).RNA Preparation and First Strand cDNA Synthesis—Total RNA from different tissues of the mice was extracted, and cDNA synthesis was performed as described previously (33.Buchmann J. Meyer C. Neschen S. Augustin R. Schmolz K. Kluge R. Al-Hasani H. Jurgens H. Eulenberg K. Wehr R. Dohrmann C. Joost H.G. Schürmann A. Endocrinology. 2007; 148: 1561-1573Crossref PubMed Scopus (68) Google Scholar). Quality of cDNA was controlled by PCR with murine glyceraldehyde-3-phosphate dehydrogenase primers (forward, 5′-ACC ACA GTC CAT GCC ATC AC-3′; reverse, 5′-TCC CAC CAC CCT GTT GCT GTA-3′).RT-PCR—For detection of deletion of exons 2–4 of Arfrp1 (Fig. 2B), reverse transcribed PCR was performed with the following primers: 5′-ATTCCGCCCGTGCTCCG-3′ and 5′-TTGATTCTGACAGCCTTTC-3′, which anneal to sequences corresponding to exon 1 and exon 5 of Arfrp1, respectively.Quantitative RT-PCR—Quantitative real time PCR analysis (qRT-PCR) was performed using the Applied Biosystems 7300 real time pcr System as described previously (33.Buchmann J. Meyer C. Neschen S. Augustin R. Schmolz K. Kluge R. Al-Hasani H. Jurgens H. Eulenberg K. Wehr R. Dohrmann C. Joost H.G. Schürmann A. Endocrinology. 2007; 148: 1561-1573Crossref PubMed Scopus (68) Google Scholar). For the determination of Arfrp1 mRNA levels in ileum and colon, a TaqMan gene expression assay was used (Arfrp1 E6_E7, Mm00513004_m1). Data were normalized (34.Livak K.J. Schmittgen T.D. Methods (San Diego). 2001; 25: 402-408Crossref PubMed Scopus (119535) Google Scholar), and a β-actin expression assay (Mm00607939_s1) was used as endogenous control.Knockdown of Endogenous ARFRP1 by shRNA Interference—The mammalian expression vector, pSUPER.basic (OligoEngine), was used for expression of shRNA targeting human ARFRP1 in HeLa cells. A gene-specific insert defining a 19-nucleotide sequence corresponding to nucleotides 691–709 (GTGGATGGTGAAGTGTGTC, GenBank™ accession number NM_003224.2, ARFRP1-shRNA) was separated by a 9-nucleotide noncomplementary loop sequence (TTCAAGAGA) from the reverse complement of the same 19-nucleotide sequence. Both sequences were subcloned into the BglII and HindIII sites of the pSUPER vector and referred to as pSUPER-ARFRP1. HeLa cells were transfected with pSUPER or pSUPER-ARFRP1 and processed for Western blot analysis or immunofluorescence after 4–8 days of incubation.Inhibition of ARFRP1 Expression by siRNA Oligonucleotides— Two small interfering RNAs (siRNA) targeting mouse ARFRP1 (GenBank™ accession number NM_029702) were used: si-a-ARFRP1 5′-GACUGUACCUGUAAGAUUGUU-3′ and si-b-RNA-ARFRP1 5′-GUAAUUGAUUCCACUGAUGUU-3′. Three control siRNAs were generated: scrambled-a 5′-GACUGUACGUGAUAGAUUGUU-3′, scrambled-b 5′-CUAAUCGAUUCCACUGAAGUU-3′, and siCONTROL™ nontargeting siRNA. The siRNAs were synthesized by Dharmacon, Inc. (Lafayette, CO). Ltk-Ecad cells were transfected with siRNAs using Lipofectamine 2000 (Invitrogen) according to manufacturer's protocols.Immunoprecipitation—MDCK-T23 cells stably expressing myc-ARFRP1 in the absence of doxycyline were incubated with ice-cold lysis buffer (10 mm imidazole, pH 6.8, 0.1 m KCl, 0.3 m sucrose, 20 μm GTP, 2 mm MgCl2, 10 mm EGTA, 1 mm NaF, 1 mm Na2MoO4, 1 mm NaVO3, 0.2% (v/v) Triton X-100, and Complete™-EDTA protease inhibitor mixture) for 10 min at 4 °C. Immunoprecipitation from precleared cell lysates (400 μg of total protein/assay) was performed with 4 μg of the anti-Myc (9E10) monoclonal antibody bound to protein-A beads as described previously (35.Weiske J. Schöneberg T. Schröder W. Hatzfeld M. Tauber R. Huber O. J. Biol. Chem. 2001; 276: 41175-41181Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 36.Weiske J. Huber O. J. Cell Sci. 2005; 118: 3117-3129Crossref PubMed Scopus (98) Google Scholar). The figure depicts a representative experiment that was repeated at least three times.Adhesion Assay—For cell aggregation assays, 105 Ltk-Ecad cells were transfected with siCONTROL™ nontargeting siRNA, ARFRP1-specific siRNAs (si-a-ARFRP1 and si-b-ARFRP1), and mutated siRNAs (scrambled-a and scrambled-b) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols. After 3 days, cell aggregation assays were performed as described previously (37.Huber O. Kermler R. Langosch D. J. Cell Sci. 1999; 112: 4415-4423Crossref PubMed Google Scholar). At the time points 0 and 45 min, two 10-μl aliquots were removed, and cells/cell aggregates were photographed using an Olympus BX-60 microscope with an Achrostigmat objective (10× magnification, 0.25 numerical aperture). JPEG images were generated with the Soft Imaging System Color View 12 and the analysis 3.0 software. After counting of particles (cells and cell aggregate), the aggregation index was calculated according to Nagafuchi and Takeichi (41.Nagafuchi A. Takeichi M. EMBO J. 1988; 7: 3679-3684Crossref PubMed Scopus (661) Google Scholar) as follows: Ai = (N0 - Nt)/N0, where N0 is the total particle number at t = 0 min, and Nt is the particle number after an incubation period of 45 min. Mean values ± S.D. of four independent aggregation assays are presented.RESULTSDistribution of E-cadherin Is Altered in Arfrp1-/-Embryos—We have previously shown that deletion of Arfrp1 in mice results in embryonic lethality because of the failure of differentiating epiblast cells to form the mesoderm. Epiblast cells of Arfrp1-/- embryos detached from the embryonic ectoderm, consistent with a defect in the regulation of cell-cell adhesion (22.Bryant D.M. Stow J.L. Trends Cell Biol. 2004; 14: 427-434Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Because changes in cadherin expression coincide with gastrulation (38.Klinowska T.C. Ireland G.W. Kimber S.J. Differentiation. 1994; 57: 7-19Crossref PubMed Scopus (19) Google Scholar), we analyzed the expression and distribution of E-cadherin and N-cadherin in control and Arfrp1-/- embryos between ED 5.0 and 6.5.In ED 5.0 control embryos (Fig. 1), a weak ARFRP1 expression is detected in ectodermal cells, and E-cadherin is located at the lateral membrane of trophectodermal epithelial cells (arrow in Fig. 1) and at the surface of ectodermal cells. In contrast, in ED 5.0 Arfrp1-/- embryos, no E-cadherin staining was visible in the trophoblast, and only a punctate pattern was detected in the embryonic ectoderm. The phenotype observed at ED 5.0 was even more pronounced at ED 6.0. At this stage, control embryos show a regular E-cadherin staining at the surface of each cell, although it is present predominantly in intracellular, aggregate-like structures in the Arfrp1-/- embryos. No N-cadherin staining was detectable in ED 5.0 and ED 6.0 embryos as described previously (data not shown, see Ref. 39.Radice G.L. Rayburn H. Matsunami H. Knudsen K.A. Takeichi M. Hynes R.O. Dev. Biol. 1997; 181: 64-78Crossref PubMed Scopus (606) Google Scholar). To test whether other plasma membrane proteins were affected in Arfrp1-/- embryos, we stained the glucose transporter GLUT1. GLUT1 was detected at the cell surface of both control and Arfrp1-/- embryos (supplemental Fig. 1A). In addition, the plasma membrane marker Na+/K+-ATPase, an integral membrane protein complex, was detected at the cell surface of the Arfrp1-/- embryos (supplemental Fig. 1B).FIGURE 1Immunohistochemical analysis of ARFRP1 and E-cadherin in control and Arfrp1-/- embryos. Serial transversal (at ED 5.0) and sagittal (at ED6.0) sections were prepared and stained with antibodies against ARFRP1 and E-cadherin as described under “Experimental Procedures.” Scale bars, 50 μm. Arrow depicts E-cadherin at the lateral membrane of trophectodermal epithelial cells. (AC, amniotic cavity; EC, embryonic ectoderm, EN, embryonic endoderm, HE, hematoxylin & eosin stain; TE, trophectoderm).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Retention of E-cadherin in the Golgi of Arfrp1-/-Intestinal Epithelial Cells—The intestinal epithelium is characterized by rapid cellular turnover with continuous proliferation, cellular migration, differentiation, and polarization (40.Zbar A.P. Simopoulos C. Karayiannakis A.J. J. Gastroenterol. 2004; 39: 413-421Crossref PubMed Scopus (50) Google Scholar). Here, E-cadherin, together with integrins, plays an important role for the development and maintenance of normal intestinal epithelial architecture and is required for complex cell-cell interactions. To analyze the role of ARFRP1 for the distribution and expression of E-cadherin in epithelial cells, we used the Cre-loxP system to disrupt Arfrp1 specifically in the intestine (Fig. 2A). Arfrp1flox/flox mice were crossed with mice carrying villin-Cre (32.Madison B.B. Dunbar L. Qiao X.T. Braunstein K. Braunstein E. Gumucio D.L. J. Biol. Chem. 2002; 277: 33275-33283Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar) to finally generate Arfrp1flox/+/vilCre/+ and Arfrp1flox/+/+/+ mice, which were intercrossed to obtain Arfrp1flox/flox/vilcre/+ mice, in the following referred to as Arfrp1vil-/- mice. Arfrp1flox/flox/+/+ mice were used as controls and referred to as Arfrp1flox/flox mice. Intestinal epithelial specific recombination of the loxP sites was confirmed by RT-PCR (Fig. 2B). In Arfrp1vil-/- mice, Arfrp1 mRNA in ileum and colon as determined by quantitative RT-PCR was reduced by 70 and 60%, respectively (Fig. 2C). Western blot analysis confirmed a similar reduction of ARFRP1 protein levels (Fig. 2D).As shown in Fig. 3, E-cadherin was localized in the lateral membrane of the cell surface of crypts and villi in control Arfrp1flox/flox mice. In contrast, in Arfrp1vil-/- mice we detected E-cadherin also in intracellular compartments (arrows in Fig. 3A) and as punctae (arrowheads in Fig. 3A) close to the plasma membrane. The expression of E-cadherin as detected by Western blotting (Fig. 3B) was not altered in ileum and colon of Arfrp1vil-/- mice.FIGURE 3Distribution and expression of E-cadherin in intestinal cells of Arfrp1flox/flox and Arfrp1vil-/- mice.A, sections of the intestine (ileum and colon) of 4-week-old Arfrp1flox/flox and Arfrp1vil-/- mice were stained with the anti-gp84 antibody against E-cadherin in combination with an Alexa488 secondary antibody, and E-cadherin distribution was analyzed by confocal laser scanning microscopy as described under “Experimental Procedures.” Arrows depict an intracellular localization of E-cadherin, and arrowheads mark the accumulation of E-cadherin in punctae within Ar" @default.
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• W2038962261 date "2008-10-01" @default.
• W2038962261 modified "2023-10-02" @default.
• W2038962261 title "ADP-ribosylation Factor-like GTPase ARFRP1 Is Required for Trans-Golgi to Plasma Membrane Trafficking of E-cadherin" @default.
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• W2038962261 doi "https://doi.org/10.1074/jbc.m802108200" @default.
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