Interaction of BP180 (Type XVII Collagen) and α6 Integrin is Necessary for Stabilization of Hemidesmosome Structure  Susan B. Hopkinson, Kirk Findlay, Gregory W. deHart, Jonathan C.R. Jones  Journal of Investigative Dermatology  Volume 111, Issue 6, Pages 1015-1022 (December 1998) DOI: 10.1046/j.1523-1747.1998.00452.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Yeast two-hybrid assays in which transfected yeast were transferred to filter paper soaked in X-gal. Inpanel 1, the transfectants coexpress a portion of BP180 (residues 1–520) and the α6 integrin. The blue color indicates binding of BP180 and α6 integrin. Inpanel 2, the blue transfectants coexpress p53 and SV40 large T-antigen, two proteins known to interact. Inpanel 3, the blue transfectants coexpress a portion of BP180 (residues 1–520) and a fragment of β4 integrin (residues 779–1566).Panel 4 illustrates yeast coexpressing a BP180 fragment (residues 1–490) and the α6 integrin. Inpanel 5, the colonies of yeast that have turned blue in the β-galactosidase assay were transfected with a BP180 cDNA, encoding residues 1–490 in vector pAS2–1, and a BP180 cDNA, encoding residues 1–520 in vector pACT.Panels 6–10 show yeast colony controls, none of which turn blue in the assay. Inpanel 6, yeast colonies express a BP180 cDNA encoding residues 1–520 and the SV40 large T-antigen cDNA in the binding domain and activating domain vectors, respectively. Inpanel 7, yeast colonies express a BP180 cDNA encoding residues 1–490 and the SV40 large T-antigen cDNA in the binding domain and activating domain vectors, respectively. Inpanel 8, the yeast colonies express p53 and α6 integrin in the binding domain and activating domain vectors, respectively. The yeast colonies inpanel 9 were transfected with the pAS2–1 binding domain vector containing a BP180 cDNA encoding residues 1–520. The colonies inpanel 10 were transfected with the pACT2 activation domain vector containing the α6 integrin insert. Journal of Investigative Dermatology 1998 111, 1015-1022DOI: (10.1046/j.1523-1747.1998.00452.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Analyses of protein–protein interaction using recombinant polypeptides. (A) SDS–PAGE analyses of recombinant α6 “light” chain (rα6LC) (lane 2)and a portion of BP180 (rBP180NCE) (lane 3). Markers indicated inlane 1 are (from bottom to top) 4, 6, 16, 30, 36, 50, 64, and 98 kDa. (B) To confirm the nature of the recombinant proteins, both rα6LC (lane 2) and rBP180NCE (lane 3) were transferred to PVDF membrane and immunoblotted with the α6 integrin monoclonal antibody 4E9G8 (lane 3) and an anti-serum directed against the BP180 peptide 180–2 (J22) (lane 3). Molecular weight standards are shown inlane 1 and are 4, 6, 16, 30, 36, 50, and 64 kDa (from bottom to top). In (C), rα6LC and rBP180NCE (lanes 1 and 2) or bovine serum albumin and rBP180NCE (lanes 3–8) were spotted individually onto separate pieces of nitrocellulose membrane as indicated. The pieces of membrane were probed with the BP180 antibody J22 and α6 integrin monoclonal antibody 4E9G8 inlanes 1 and 2, respectively, to confirm protein absorption to the nitrocellulose.Lanes 3–5 and 7–8 were overlaid with rα6LC and then subsequently processed with antibody 4E9G8.Lane 3 was overlaid with rα6LC alone.Lane 4 was incubated with rα6LC in the presence of 100 μg peptide 180–2 per ml, whereaslane 5 was overlaid with rα6LC in the presence of 100 μg 180-SCR peptide per ml.Lanes 7 and 8 were overlaid with rα6LC in the presence of normal rabbit IgG or J22 anti-serum (1:20 dilution), respectively. Note the strong 4E9G8 antibody reactivity inlanes 3, 5, and 7. The diminished reactivity inlanes 4 and 8 indicates that peptide 180–2 as well as an anti-serum against the same peptide inhibit rBP180NCE/rα6LC interaction.Lane 6 shows a control where the nitrocellulose piece was processed for overlay but rα6LC was omitted from the overlay buffer. This confirms that there is no reactivity of rBP180NCE inlane 6 with antibody probe 4E9G8. The results of coimmunoprecipitation studies using rBP180NCE and rα6LC are shown in (D). In the case oflanes 1–3, ≈1 mg of recombinant rBP180NCE and α6 integrin were mixed overnight at 4°C. rα6LC used inlane 2 was preincubated for 4 h at 4°C with 1 mg 180–2 peptide per ml prior to addition of rBP180NCE, whereas that inlane 3 was preincubated with 1 mg control peptide per ml. Following overnight incubation, a6 antibody 4E9G8 was added to the mix at a 1:50 dilution, followed by protein G beads. The precipitated proteins were subsequently processed for western blotting using either 4E9G8 antibody to detect precipitated rα6LC (upper panel) or a rBP180NCE probe as indicated (lower panel). Equal amounts of rα6LC were precipitated inlanes 1–3 as determined by scanning densitometry. rBP180NCE was coprecipitated inlanes 1–3, but there is a reduced quantity in the sample in which rα6LC had been preincubated with 180–2 peptide (lane 2).Lane 4 shows a control precipitate in which rα6LC was omitted. This reveals that rBP180NCE is not precipitated nonspecifically by the 4E9G8 antibodies. Journal of Investigative Dermatology 1998 111, 1015-1022DOI: (10.1046/j.1523-1747.1998.00452.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunofluorescence analyses of 804G cells plated onto coverslips in medium containing 0.5 mM 180-SCR peptide (A–C) or medium containing 0.5 mM 180–2 peptide (D–F). After 24 h the coverslips were prepared for double immunofluorescence using BP230 antibody 5E (A, D) and BP180 anti-serum J17 (B, E). Phase images of the cells are seen in (C) and (F). In (A) and (B), BP230 and BP180 colocalize in a Swiss cheese pattern, a localization that indicates the presence of mature hemidesmosomes. Although BP230 and BP180 show primarily a basal codistribution, there is no obvious Swiss cheese pattern in (D) and (E).Scale bar: 20 μm. Journal of Investigative Dermatology 1998 111, 1015-1022DOI: (10.1046/j.1523-1747.1998.00452.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunofluorescence analyses of 804G cells plated into medium containing 180-SCR (A–C) or 180–2 (D–F() peptide at a concentration of 0.5 mM, and 24 h later processed for immunofluorescence using α6 integrin antibodies (A, D) and BP230 antibody 5EB, E). In (A) and (B), the antibodies colocalize in a Swiss cheese pattern. In the insets to (A) and (B), z-sections of the cells are shown and indicate the primarily basal localization of both BP230 and α6 integrin. The plane of the section is indicated by thesmall arrow. In contrast, 24 h after plating 804G cells into medium containing 180–2 peptide, α6 integrin (D) and BP230 (E) show only a limited area of costaining in a Swiss cheese pattern (arrowhead). More importantly, in the regionsarrowed in (D) and (E) towards the periphery of the cells, α6 integrin is found in the absence of any obvious BP230 staining. This is even more obvious in the z-sections shown in the insets of (D) and (E). The plane of section is indicated by thesmall arrow. Phase contrast images of the cells are shown in (C) and (F).Scale bar: 20 μm. Journal of Investigative Dermatology 1998 111, 1015-1022DOI: (10.1046/j.1523-1747.1998.00452.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Immunofluorescence analyses of 804G cells plated into medium containing 180-SCR (A–C) or 180–2 (D–F) peptide at a concentration of 0.5 mM and 24 h later processed for immunofluorescence using α6 integrin antibodies (A, D() and a mouse monoclonal antibody against BP180B, E).In (A) and (B), the antibodies colocalize in a Swiss cheese pattern. In the insets to (A) and (B), z-sections of the cells are shown and indicate the mostly basal localization of both α6 integrin and BP180. The plane of the section is indicated by thesmall arrow. In contrast, 24 h after plating 804G cells into medium containing 180–2 peptide, α6 integrin is distributed primaily at the cell edge (D), whereas BP180 is located in spots towards the cell center in the region where the cells interact with the substratum (E). In the insets to (D) and (E), z-sections of the cells are shown. The plane of the section is indicated by thesmall arrow in (D) and (E). In the insets, note that thearrowhead indicates a region where α6 integrin and BP180 colocalize towards the center of the cell, whereas thearrow marks α6 localization at the edge of the cell, in the absence of any obvious BP180 staining.Scale bar: 20 μm. Journal of Investigative Dermatology 1998 111, 1015-1022DOI: (10.1046/j.1523-1747.1998.00452.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Ultrastructural analyses of peptide-treated 804G cells. 804G cells grown in culture for 24 h in the presence of 0.5 mM 180-SCR (A) or 180–2 peptide (B) were fixed and processed for electron microscopy. In (A), three hemidesmosomes are observed along the region of cell–substrate interface (arrows). These possess cytoplasmic plaques and subbasal dense plates in the extracellular space. At the site of the hemidesmosome at the left of the image, individual filaments emerge from a large keratin bundle and associate with the most cytoplasmic region of the hemidesmosome plaque. Cells incubated in medium containing 180–2 peptide for 24 h possess few mature hemidesmosomes (B). This is consistent with the disruption of hemidesmosome protein localization observed in similarly treated cells processed for immunofluorescence evaluation (seeFigs 3–5).Scale bar: 300 nm. Journal of Investigative Dermatology 1998 111, 1015-1022DOI: (10.1046/j.1523-1747.1998.00452.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions