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• W2010063139 abstract "The hemidesmosome is a multimolecular complex that integrates the extracellular matrix with the keratin cytoskeleton and that stabilizes epithelial attachment to connective tissue. A 180 kDa protein (BP180, type XVII collagen), first identified by its reactivity with autoantibodies in the serum of patients with a blistering skin disease called bullous pemphigoid (BP), is a transmembrane component of the hemidesmosome with a collagen-like extracellular domain. Here, using recombinantly expressed molecules and the yeast two-hybrid assay, we have identified α6 integrin as a BP180-binding partner. The association between specific domains of the BP180 and α6 integrin molecules is inhibited by a 14 mer peptide, whose sequence is identical to amino acid residues 506–519 in the noncollagenous region of the ectodomain of the BP180 molecule, as well as by antibodies raised against this peptide. The 14 mer peptide sequence is part of an epitope recognized by autoantibodies that are pathogenic in BP.In vivo, when 804G cells are plated into medium containing the same peptide, they fail to assemble hemidesmosomes. Furthermore, although BP180 and certain cytoplasmic components of the hemidesmosome colocalize in the peptide-treated cells, they are aberrantly distributed and fail to show extensive association with α6β4 integrin. Taken together, our results indicate that BP180 is a novel transmembrane ligand of the α6β4 integrin heterodimer. In addition, our data provide support for the possibility that BP180 and α6 integrin interaction is not only mediated by the BP epitope but is necessary for hemidesmosome formation. The hemidesmosome is a multimolecular complex that integrates the extracellular matrix with the keratin cytoskeleton and that stabilizes epithelial attachment to connective tissue. A 180 kDa protein (BP180, type XVII collagen), first identified by its reactivity with autoantibodies in the serum of patients with a blistering skin disease called bullous pemphigoid (BP), is a transmembrane component of the hemidesmosome with a collagen-like extracellular domain. Here, using recombinantly expressed molecules and the yeast two-hybrid assay, we have identified α6 integrin as a BP180-binding partner. The association between specific domains of the BP180 and α6 integrin molecules is inhibited by a 14 mer peptide, whose sequence is identical to amino acid residues 506–519 in the noncollagenous region of the ectodomain of the BP180 molecule, as well as by antibodies raised against this peptide. The 14 mer peptide sequence is part of an epitope recognized by autoantibodies that are pathogenic in BP.In vivo, when 804G cells are plated into medium containing the same peptide, they fail to assemble hemidesmosomes. Furthermore, although BP180 and certain cytoplasmic components of the hemidesmosome colocalize in the peptide-treated cells, they are aberrantly distributed and fail to show extensive association with α6β4 integrin. Taken together, our results indicate that BP180 is a novel transmembrane ligand of the α6β4 integrin heterodimer. In addition, our data provide support for the possibility that BP180 and α6 integrin interaction is not only mediated by the BP epitope but is necessary for hemidesmosome formation. bullous pemphigoid herpes gestationis noncollagenous region of the ectodomain The major function of an integrin is as a receptor for extracellular matrix molecules (Hynes, 1992Hynes R.O. Integrins: versatility, modulation, and signaling in cell adhesion.Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8815) Google Scholar). In addition, integrins mediate signals from matrix to cells and vice versa (Hynes, 1992Hynes R.O. Integrins: versatility, modulation, and signaling in cell adhesion.Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8815) Google Scholar;Clark and Brugge, 1995Clark E.A. Brugge J.S. Integrins and signal transduction pathways, the road taken.Science. 1995; 268: 233-239Crossref PubMed Scopus (2758) Google Scholar;Dedhar and Hannigan, 1996Dedhar S. Hannigan G.E. Integrin cytoplasmic interactions and bidrectional transmembrane signalling.Curr Op Cell Biol. 1996; 8: 657-669Crossref PubMed Scopus (341) Google Scholar). The α6β4 integrin heterodimer is no exception because there is evidence that it binds laminin-5, and its β subunit interacts with various proteins involved in signaling events (Marchisio et al., 1993Marchisio P.C. Cremona O. Savoia P. et al.The basement membrane protein BM-600/nicein codistributes with kalinin and the integrin alpha 6 beta 4 in human cultured keratinocytes.Exp Cell Res. 1993; 205: 205-212Crossref PubMed Scopus (47) Google Scholar;Mainiero et al., 1995Mainiero F. Pepe A. Wary K.K. Spinardi L. Mohammadi M. Schlessinger J. Giancotti F.G. Signal transduction by the α6β4 integrin: distinct β;4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes.Embo. 1995; 14: 4470-4481PubMed Google Scholar;Spinardi et al., 1995Spinardi L. Einheber S. Cullen T. Milner T.A. Giancotti F.G. Recombinant tail-less integrin β4 subunit disrupts hemidesmosomes, but does not suppress α6β;4 -mediated cell adhesion to laminins.J Cell Biol. 1995; 129: 473-487Crossref PubMed Scopus (152) Google Scholar;Giancotti, 1996Giancotti F.G. Signal transduction by the alpha 6 beta 4 integrin: charting the path between laminin binding and nuclear events.J Cell Sci. 1996; 109: 1165-1172PubMed Google Scholar;Green and Jones, 1996Green K.J. Jones JcR Desmosomes and hemidesmosomes: structure and function of molecular components.Faseb J. 1996; 10: 871-880Crossref PubMed Scopus (289) Google Scholar); however, the α6β4 heterodimer subunit is unusual because it is found in an epithelial cell–matrix junction, called the hemidesmosome, which tethers the keratin rather than the microfilament cytoskeleton to the cell surface (Stepp et al., 1990Stepp M.A. Spurr-Michaud S. Tisdale A. Elwell J. Gipson I.K. Alpha 6 beta 4 integrin heterodimer is a component of hemidesmosomes.Proc Natl Acad Sci USA. 1990; 87: 8970-8974Crossref PubMed Scopus (429) Google Scholar;Jones et al., 1991Jones Jcr Kurpakus M.A. Cooper H.M. Quaranta V.A. function for the integrin alpha 6 beta 4 in the hemidesmosome.Cell Reg. 1991; 2: 427-438Crossref PubMed Scopus (222) Google Scholar;Quaranta and Jones, 1991Quaranta V. Jones JcR The internal affairs of an integrin.Trends Cell Biol. 1991; 1: 2-4Abstract Full Text PDF PubMed Scopus (62) Google Scholar;Sonnenberg et al., 1991Sonnenberg A. Calafat J. Janssen H. et al.Integrin α6/β;4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion.J Cell Biol. 1991; 113: 907-917Crossref PubMed Scopus (490) Google Scholar;Green and Jones, 1996Green K.J. Jones JcR Desmosomes and hemidesmosomes: structure and function of molecular components.Faseb J. 1996; 10: 871-880Crossref PubMed Scopus (289) Google Scholar). The hemidesmosome is a complex structure, befitting its roles in cytoskeleton attachment, matrix adhesion, and signal transduction (Mainiero et al., 1995Mainiero F. Pepe A. Wary K.K. Spinardi L. Mohammadi M. Schlessinger J. Giancotti F.G. Signal transduction by the α6β4 integrin: distinct β;4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes.Embo. 1995; 14: 4470-4481PubMed Google Scholar;Jones et al., 1994Jones Jcr Asmuth J. Baker S.E. Langhofer M. Roth S.I. Hopkinson S.B. Hemidesmosomes: extracellular matrix/intermediate filament connectors.Exp Cell Res. 1994; 213: 1-11Crossref PubMed Scopus (88) Google Scholar;Baker et al., 1996Baker S.E. Hopkinson S.B. Fitchmun M. et al.Laminin-5 and hemidesmosomes: role of the α3 chain subunit in hemidesmosome stability and assembly.J Cell Sci. 1996; 109: 2509-2520Crossref PubMed Google Scholar;Giancotti, 1996Giancotti F.G. Signal transduction by the alpha 6 beta 4 integrin: charting the path between laminin binding and nuclear events.J Cell Sci. 1996; 109: 1165-1172PubMed Google Scholar;Green and Jones, 1996Green K.J. Jones JcR Desmosomes and hemidesmosomes: structure and function of molecular components.Faseb J. 1996; 10: 871-880Crossref PubMed Scopus (289) Google Scholar). Two well-studied hemidesmosome proteins are human autoantigens of 230 kDa (BP230, BPAG1) and 180 kDa (BP180, BPAG2, type XVII collagen). These proteins were identified using autoantibodies in the sera of patients with bullous pemphigoid (BP), a blistering skin disease (Klatte et al., 1989Klatte D.H. Kurpakus M.A. Grelling K.A. Jones JcR Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells.J Cell Biol. 1989; 109: 3377-3390Crossref PubMed Scopus (116) Google Scholar;Stanley, 1993Stanley J.R. Cell adhesion molecules as targets of autoanitbodies in pemphigus and pemphigoid, bullous diseases due to epidermal cell adhesion.Adv Immunol. 1993; 53: 291-325Crossref PubMed Scopus (223) Google Scholar). BP230, a hemidesmosome cytoplasmic plaque component, plays a role in anchorage of keratin filaments to the cell surface (Guo et al., 1995Guo L. Degenstein L. Dowling J. Yu Q.-C. Wollmann R. Perman B. Fuchs E. Gene targeting of BPAG1: abnormalities in mechanical strength and cell migration in stratified squamous epithelia and neurologic degeneration.Cell. 1995; 81: 233-243Abstract Full Text PDF PubMed Scopus (387) Google Scholar). In contrast, BP180 is a type II transmembrane protein with a 55 kDa cytoplasmic domain and an extracellular region that is composed of a series of collagen-like repeats, thought to interact with other basement membrane molecules (Giudice et al., 1991aGiudice G.J. Emery D.J. Diaz L.A. Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180.J Invest Dermatol. 1991 a; 99: 243-250Crossref Scopus (469) Google Scholar, b 1993;Hopkinson et al., 1992Hopkinson S.B. Riddelle K.S. Jones JcR Cyoplasmic domain of the 180-kD bullous pemphigoid antigen, a hemidesmosomal component: molecular and cell biologic characterization.J Invest Dermatol. 1992; 99: 264-270Crossref PubMed Scopus (123) Google Scholar,Hopkinson et al., 1996Hopkinson S.B. Jones Jcr Collagen XviI and collagen related molecules: linked by more than a common motif.Sem Cell Dev Biol. 1996; 7: 659-666Crossref Scopus (3) Google Scholar;Liu et al., 1993Liu Z. Diaz L.A. Troy J.L. Taylor A.F. Emery D.J. Fairley J.A. Giudice G.J. A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen BP180.J Clin Invest. 1993; 92: 2480-2488Crossref PubMed Scopus (529) Google Scholar). To elucidate roles for the noncollagenous regions of BP180 in targeting the molecule to its correct cellular location as well as mediating its protein associations, we previously undertook molecular genetic analyses of various domains of the BP180 molecule (Hopkinson et al., 1995Hopkinson S.B. Baker S.E. Jones J.C.R. Molecular genetic studies of a human epidermal autoantigen (the 180kD bullous pemphigoid antigen/BP180): identification of functionally important sequences within the BP180 molecule and evidence for an interaction between BP180 and α6 integrin.J Cell Biol. 1995; 130: 117-126Crossref PubMed Scopus (111) Google Scholar). We found that the N-terminal 36 residues of BP180 are essential for polarization of the molecule to the basal surface, whereas 27 extracellular residues immediately following the membrane spanning domain of BP180 appear to mediate BP180 interactions with other hemidesmosome proteins (Hopkinson et al., 1995Hopkinson S.B. Baker S.E. Jones J.C.R. Molecular genetic studies of a human epidermal autoantigen (the 180kD bullous pemphigoid antigen/BP180): identification of functionally important sequences within the BP180 molecule and evidence for an interaction between BP180 and α6 integrin.J Cell Biol. 1995; 130: 117-126Crossref PubMed Scopus (111) Google Scholar,Hopkinson et al., 1996Hopkinson S.B. Jones Jcr Collagen XviI and collagen related molecules: linked by more than a common motif.Sem Cell Dev Biol. 1996; 7: 659-666Crossref Scopus (3) Google Scholar). Intriguingly, the same region contains the epitope of pathogenic BP autoantibodies as well as that of autoantibodies in another blistering disease, herpes gestationis (HG) (Giudice et al., 1993Giudice G.J. Emery D.J. Zelickson B.D. Anhalt G.J. Liu Z. Diaz L.A. Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain.J Immunol. 1993; 151: 5742-5750PubMed Google Scholar). Using yeast two-hybrid assays, BP180 peptides, andin vitro assays of protein binding, we show that specific domains of BP180 and α6 integrin can interact directly and that the sequence containing the pathogenic autoantibody epitope in BP180 is most likely involved in this interaction. Moreover, peptides based on this sequence disrupt hemidesmosome protein localization and hemidesmosome assembly in living cells. 804G cells were cultured as detailed previously (Riddelle et al., 1991Riddelle K.S. Green K.J. Jones J.C.R. Formation of hemidesmosomes in vitro by a rat bladder cell line.J Cell Biol. 1991; 112: 159-168Crossref PubMed Scopus (73) Google Scholar). In some experiments, 804G cells were plated into medium containing peptides at a concentration of 0.5 mM and incubated overnight. In brief, two BP180 cDNA, encoding amino acid residues 1–520 and 1–490 were ligated separately into the DNA-binding domain cloning vector pAS2–1 (Clontech, Palo Alto, CA). The first of these encodes the entire BP180 cytoplasmic domain, the membrane spanning region, as well as a portion of the noncollagenous region of the ectodomain (NCE) that includes the BP180 autoantibody epitope. The second encodes the BP180 cytoplasmic domain and the membrane spanning region of the molecule but lacks the NCE domain. A full-length α6 integrin cDNA (a gift from Dr. Vito Quaranta, Scripps Research Institute, La Jolla, CA), a β4 integrin cDNA, encoding residues 779–1566 of the cytoplasmic domain of the molecule, and a BP180 cDNA, encoding amino acid residues 1–520, were ligated separately into the DNA activation domain cloning vector pACT2 (Clontech). All constructs were sequenced through the vector/insert ligation region to ensure that the cDNA were in frame. Individual clones of constructs were grown in selective media and DNA prepared from the clones using a Qiagen Maxi kit (Qiagen, Chatsworth, CA). The DNA preparations were then used to transform the yeast strain Y190, according to protocols outlined in the Matchmaker Two-Hybrid System 2 Manual (Clontech). Transfected colonies were selected by growth in medium lacking leucine, tryptophan, and histidine but containing 25 mM 3-amino-1,2,4-triazole. The latter was used to inhibit low levels of “leaky” expression of His3p in the reporter yeast strain. The resulting yeast colonies were spotted onto nylon filters and flash frozen in liquid nitrogen. To detect activation of the reporter gene lacZ and resulting expression of β-galactosidase, the filters were placed on Whatman paper soaked in a solution containing X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). A binding domain plasmid (pVA3–1), containing a cDNA encoding murine p53, and an activation domain plasmid (pTD1–1), containing the SV40 large T-antigen coding sequence, were used as part of our control studies (Clontech). Specific domains of both α6 integrin and BP180 were prepared recombinantly using an expression system in which polypeptides, fused with a 6xHis affinity tag, are expressed inE. coli (Qiagen). To prepare the relevant portion of BP180, polymerase chain reaction was performed on a BP180 cDNA using a 22 bp primer beginning with sequence corresponding to the first putative extracellular residue of the NCE domain and a second 22 bp primer that corresponds to a sequence located just before the start of the first collagen region of the molecule (Giudice et al., 1991bGiudice G.J. Squiquera H.L. Elias P.M. Diaz L.A. Identification of two collagen domains within the bullous pemphigoid autoantigen, BP180.J Clin Invest. 1991 b; 87: 734-738Crossref PubMed Scopus (97) Google Scholar;Hopkinson et al., 1992Hopkinson S.B. Riddelle K.S. Jones JcR Cyoplasmic domain of the 180-kD bullous pemphigoid antigen, a hemidesmosomal component: molecular and cell biologic characterization.J Invest Dermatol. 1992; 99: 264-270Crossref PubMed Scopus (123) Google Scholar). This fragment was subcloned in-frame into a Qiagen expression vector. Similarly, an α6 integrin fragment was generated by polymerase chain reaction using primers that flank the sequence encoding the “light” chain of the molecule (Tamura et al., 1990Tamura R.N. Rozzo C. Starr L. Chambers J. Reichardt L.F. Cooper H. Quaranta V. Epithelial integrin alpha 6 beta 4: complete primary structure of alpha 6 and variant forms of beta 4.J Cell Biol. 1990; 111: 1593-1604Crossref PubMed Scopus (259) Google Scholar). The resultant polymerase chain reaction product was also subcloned into a Qiagen vector and transfected intoE. coli cells that were induced to produce protein with isopropyl-β-D-thiogalactopyranoside. The cells were lyzed and extracts were incubated overnight in a 6 M urea buffer. Cell extracts were passed over a His.Bind Resin column (Novagen, Madison, WI) and bound fusion protein eluted in an imidazole elution buffer in the presence of 6 M urea. The eluent was dialyzed against 10 mM tris buffer (pH 7.5) overnight at 4°C, concentrated by lyophilization, and resuspended in sterile H2O. The purity of the recombinant polypeptides was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/immunoblotting. A peptide (180–2) (RSILPYGDSMDRIE) corresponding to amino acid residues 506–519 of BP180 and a peptide (180-SCR) (DLGMISPRDEIYSR) of identical amino acid composition in which the amino acids have been scrambled, were prepared by Bio-Synthesis (Lewisville, TX). The former peptide lies in the region of the NCE domain, the target site of human autoantibodies in BP and HG (Giudice et al., 1993Giudice G.J. Emery D.J. Zelickson B.D. Anhalt G.J. Liu Z. Diaz L.A. Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain.J Immunol. 1993; 151: 5742-5750PubMed Google Scholar). A peptide corresponding to residues 310–323 (180-CYTO) (KNMPQSPAAVNTGV) in the cytoplasmic domain was also prepared. Peptides were dissolved directly in culture medium or in the appropriate buffer (see below), respectively. 5E, a human monoclonal antibody against BP230, was a gift from Dr. Takashi Hashimoto (Keio University, Tokyo, Japan) (Hashimoto et al., 1993Hashimoto T. Amagai M. Ebihara T. et al.Further analyses of epitopes for human monoclonal anti-basement membrane zone antibodies produced by stable human hybridoma cell lines constructed with Epstein–Barr virus transformants.J Invest Dermatol. 1993; 100: 310-315Abstract Full Text PDF PubMed Google Scholar). Affinity-purified rabbit antibodies against α6A integrin were provided by Dr. Vito Quaranta (Tamura et al., 1990Tamura R.N. Rozzo C. Starr L. Chambers J. Reichardt L.F. Cooper H. Quaranta V. Epithelial integrin alpha 6 beta 4: complete primary structure of alpha 6 and variant forms of beta 4.J Cell Biol. 1990; 111: 1593-1604Crossref PubMed Scopus (259) Google Scholar). GoH3, a rat monoclonal antibody against the α6 integrin subunit, and mouse monoclonal antibody 4E9G8, against the cytoplasmic domain of the α6A integrin light chain subunit, were purchased from Immunotech (Westbrook, ME). J17 anti-serum and a mouse monoclonal antibody against the N-terminal domain of BP180 were described inHopkinson et al., 1992Hopkinson S.B. Riddelle K.S. Jones JcR Cyoplasmic domain of the 180-kD bullous pemphigoid antigen, a hemidesmosomal component: molecular and cell biologic characterization.J Invest Dermatol. 1992; 99: 264-270Crossref PubMed Scopus (123) Google Scholar andRiddelle et al., 1992Riddelle K.S. Hopkinson S.B. Jones J.C.R. Hemidesmosomes in the cultured cell line 804G: their fate during wound closure, mitosis and drug induced alterations of the cytoskeleton.J Cell Sci. 1992; 103: 475-490PubMed Google Scholar. Anti-serum J22 was prepared against peptide 180–2 in the following manner. The peptide was conjugated to keyhole limpet hemocyanin using a kit (Pierce, Rockford, IL). This conjugate was injected into a rabbit as detailed previously (Hopkinson et al., 1992Hopkinson S.B. Riddelle K.S. Jones JcR Cyoplasmic domain of the 180-kD bullous pemphigoid antigen, a hemidesmosomal component: molecular and cell biologic characterization.J Invest Dermatol. 1992; 99: 264-270Crossref PubMed Scopus (123) Google Scholar). Serum was collected at monthly intervals and tested for reactivity against 180–2 peptide, which was spotted onto strips of nitrocellulose. Recombinant proteins, solubilized in Laemmli sample buffer, were subjected to SDS-PAGE using 4%–20% acrylamide gels (Bio-Rad, Hercules, CA) (Laemmli, 1970Laemmli U.K. Cleavage of structural proteins during assembly of the head of bacteriophage T4.Nature (Lond). 1970; 277: 680-685Crossref Scopus (202763) Google Scholar) The gels were stained using the BioRad silver stain protocol In addition, proteins separated on gels were transferred to PVDF membrane for immunoblot analyses Alkaline phosphatase secondary antibodies, purchased from Jackson Immunoresearch (West Grove, PA), were used to detect bound primary antibody and immunoblots were developed using a phosphatase development kit (Sigma, St Louis, MO) For immunoprecipitation studies, '1 μg each of the recombinant α6 integrin and BP180 fragments were mixed together in 250 μl of tris buffered-saline (pH 80) containing 1 mM MgCl2 and 2 mM CaCl2 After overnight incubation at 4°C, 4E9G8 α6 antibody was added to a 1:50 dilution and incubated at 4°C for a further 2 h Subsequently, 20 μl of protein G agarose (Gibco/BRL, Gaithersburg, MD) were added to the mix for an additional 2 h The protein G agarose was collected by centrifugation, washed four times in buffer, and then boiled in SDS-PAGE sample buffer The resulting protein solution was processed for western immunoblotting as detailed above using either an α6 integrin or a BP180 probe The western immunoblots were scanned densitometrically and then analyzed using Bio-Rad Molecular Analyst software (Bio-Rad, Hercules, CA) For blot overlay assays, recombinant protein was spotted onto nitrocellulose at an approximate concentration of 0.1 μg per ml. The spots were allowed to dry and then the nitrocellulose was blocked in overlay buffer. Protein solutions with and without peptides were overlaid on the nitrocelluose for 2 h at room temperature. Following washing in overlay buffer, the nitrocellulose was incubated in primary antibody to detect any bound protein. The nitrocellulose was further incubated in secondary alkaline phosphatase antibody and developed using a kit (Sigma). Cells, grown on glass coverslips, were fixed for 30 s in 3.7% formaldehyde and then extracted in 0.5% Triton X-100 at 4°C for 8 min. Double label immunofluorescence was performed as detailed inRiddelle et al., 1991Riddelle K.S. Green K.J. Jones J.C.R. Formation of hemidesmosomes in vitro by a rat bladder cell line.J Cell Biol. 1991; 112: 159-168Crossref PubMed Scopus (73) Google Scholar. After mounting, coverslips were viewed on a Zeiss LSM10 confocal microscope (Carl Zeiss, Thornwood, NY). Controls for immunocytochemistry included omission of primary antibodies or use of irrelevant IgG to determine nonspecific binding of secondary antibodies. Fixing and processing of cells for electron microscopy was performed as described inRiddelle et al., 1991Riddelle K.S. Green K.J. Jones J.C.R. Formation of hemidesmosomes in vitro by a rat bladder cell line.J Cell Biol. 1991; 112: 159-168Crossref PubMed Scopus (73) Google Scholar. Sections were cut perpendicular to the substratum and viewed on a JEOL (Peabody, MA) 100CX microscope or 1220 electron microscope at 60 kV. Morphometric analyses were undertaken using JEOL software. We previously showed that a fragment of BP180, consisting of its cytoplasmic and transmembane domain as well as 30 extracellular residues of the NCE domain, is capable of associating with other hemidesmosome elements along regions of cell–matrix interaction in 804G cells (Hopkinson et al., 1995Hopkinson S.B. Baker S.E. Jones J.C.R. Molecular genetic studies of a human epidermal autoantigen (the 180kD bullous pemphigoid antigen/BP180): identification of functionally important sequences within the BP180 molecule and evidence for an interaction between BP180 and α6 integrin.J Cell Biol. 1995; 130: 117-126Crossref PubMed Scopus (111) Google Scholar). Furthermore, we have speculated that BP180 may bind the α6 integrin subunit of the hemidesmosome, via an interaction between the NCE domain of BP180 and the extracellular domain of α6 integrin (Hopkinson et al., 1995Hopkinson S.B. Baker S.E. Jones J.C.R. Molecular genetic studies of a human epidermal autoantigen (the 180kD bullous pemphigoid antigen/BP180): identification of functionally important sequences within the BP180 molecule and evidence for an interaction between BP180 and α6 integrin.J Cell Biol. 1995; 130: 117-126Crossref PubMed Scopus (111) Google Scholar). To provide direct evidence for such an association, we used the yeast two-hybrid system. In initial studies, we expressed a cDNA that encodes part of BP180, namely, residues 1–520 that include the BP180 autoantibody epitope, and α6 integrin cDNA in the DNA-binding domain of the pAS2–1 cloning vector and the DNA activation domain of the pACT2 cloning vector, respectively, as detailed inMaterials and Methods. These vectors were cotransfected into yeast that were then plated onto plates lacking leucine, tryptophan, and histidine. The transfected yeast grow, implying an interaction between BP180 and α6 integrin. In addition, in the two-hybrid system, interaction between the two test proteins is necessary for the activation of transcription of the lacZ reporter gene in transfected yeast clones (Fields and Sternglanz, 1994Fields S. Sternglanz R. The two hybrid system: an assay for protein–protein interactions.Trends Genet. 1994; 10: 286-292Abstract Full Text PDF PubMed Scopus (506) Google Scholar). The expression of the gene can be assessed using a blue/white β-galactosidase assay. Yeast cotransfected with plasmids containing a BP180 cDNA encoding residues 1–520 together with α6 integrin vectors and transferred to X-gal soaked filters become blue in color, as do yeast cotransfected with the plasmids pVA3–1 and pTD1–1 containing the coding sequences of p53 and large T-antigen, two proteins whose interaction has been shown in this systemFigure 1,panels 1 and 2;Li and Fields, 1993Li B. Fields S. Identification of mutations in p53 that affect its binding to SV40 large T antigen by using the yeast two-hybrid system.Faseb J. 1993; 7: 957-963Crossref PubMed Scopus (188) Google Scholar;Li et al., 1993Li K. Tamai K. Tan Eml Uitto J. Cloning of type XVII collagen, complementary and genomic DNA sequences of mouse 180-kilodalton bullous pemphigoid antigen (BPAG2) predict an interrupted collagenous domain, a transmembrane segment, and unusual features in the 5′-end of the gene and the 3′-untranslated region of the mRNA.J Biol Chem. 1993; 268: 8825-8834Abstract Full Text PDF PubMed Google Scholar). In addition, yeast cotransfected with the same BP180 cDNA, encoding residues 1–520 in the pAS2–1 vector, and vector pACT containing a cDNA encoding residues 779–1566 of the β4 integrin cytoaplsmic domain, also turn blue in the β-galactosidase assay (Figure 1,panel 3). This indicates that BP180 and α6 integrin can interact and confirms the results of others that BP180 can bind the cytoplasmic domain of β4 integrin (Aho and Uitto, 1998Aho S. Uitto J. Direct interacttion between the intracellular domains of bullous pemphigoid antigen 2 (BP180) and β;4 integrin, hemidesmosomal components of basal keratinocytes.Biochem Biophys Res Comm. 1998; 243: 694-699Crossref PubMed Scopus (55) Google Scholar;Schaapveld et al., 1998Schaapveld Rqj Borradori L. Geerts D. et al.Hemidesmosome formation is initiated by the β4 integrin subunit, requires complex formation of β4 and HD1 /plectin, and involves a direct interaction between β;4 and the bullous pemphigoid antigen BP180.J Cell Biol. 1998; 142: 271-284Crossref PubMed Scopus (148) Google Scholar). In contrast, no color change was observed in yeast colonies that were cotransfected with the pAS2–1 vector containing a BP180 cDNA, encoding residues 1–490 (i.e., lacking the BP extracellular autoantibody epitope), and α6 integrin in the pACT2 cloning vector (Figure 1,panel 4). To ensure that the BP180 fragment encoded by the former cDNA is capable of entering the nuclei of yeast and interacting with other proteins, yeast were transfected with this cDNA in the pAS2–1 vector together with a pACT2 vector containing the cDNA encoding residues 1–520 of BP180. The transfected yeast grow on restrictive media and turn blue in a β-galactosidase assay (Figure 1,panel 5). Taken together, these results indicate that the BP extracellular autoantibody epitope is involved in the association of BP180 and α6 integrin. No blue colonies were observed in any of the controls shown inFigure 1. These included assays of yeast cotransfected with BP180 cDNA, encoding residues 1–520 and 1–490, in vector pAS2–1 together with the plasmid pTD1–1 (Figure 1,panels 6 and 7; result not shown), yeast cotransfected with plasmid pVA3–1 and α6 integrin in vector pACT2 (Figure 1,panel 8), yeast colonies transfected with BP180 cDNA, encoding residues 1–520, in either vector pAS2–1 or vector pACT (Figure 1,panel 9), yeast colonies transfected with BP180 cDNA, encoding residues 1–490, in vector pAS2–1 (result not shown), as wel" @default.
• W2010063139 created "2016-06-24" @default.
• W2010063139 creator A5016828880 @default.
• W2010063139 creator A5035402807 @default.
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• W2010063139 date "1998-12-01" @default.
• W2010063139 modified "2023-10-15" @default.
• W2010063139 title "Interaction of BP180 (Type XVII Collagen) and α6 Integrin is Necessary for Stabilization of Hemidesmosome Structure" @default.
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