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, 136 (6), 1333-47

The Localization of Bullous Pemphigoid Antigen 180 (BP180) in Hemidesmosomes Is Mediated by Its Cytoplasmic Domain and Seems to Be Regulated by the beta4 Integrin Subunit

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The Localization of Bullous Pemphigoid Antigen 180 (BP180) in Hemidesmosomes Is Mediated by Its Cytoplasmic Domain and Seems to Be Regulated by the beta4 Integrin Subunit

L Borradori et al. J Cell Biol.

Abstract

Bullous pemphigoid antigen 180 (BP180) is a component of hemidesmosomes, i.e., cell-substrate adhesion complexes. To determine the function of specific sequences of BP180 to its incorporation in hemidesmosomes, we have transfected 804G cells with cDNA-constructs encoding wild-type and deletion mutant forms of human BP180. The results show that the cytoplasmic domain of BP180 contains sufficient information for the recruitment of the protein into hemidesmosomes because removal of the extracellular and transmembrane domains does not abolish targeting. Expression of chimeric proteins, which consist of the membrane targeting sequence of K-Ras fused to the cytoplasmic domain of BP180 with increasing internal deletions or lacking the NH2 terminus, indicates that the localization of BP180 in hemidesmosomes is mediated by a segment that spans 265 amino acids. This segment comprises two important regions located within the central part and at the NH2 terminus of the cytoplasmic domain of BP180. To investigate the effect of the alpha6beta4 integrin on the subcellular distribution of BP180, we have transfected COS-7 cells, which lack alpha6beta4 and BP180, with cDNAs for BP180 as well as for human alpha6A and beta4. We provide evidence that a mutant form of BP180 lacking the collagenous extracellular domain as well as a chimeric protein, which contains the entire cytoplasmic domain of BP180, are colocalized with alpha6beta4. In contrast, when cells were transfected with cDNAs for alpha6A and mutant forms of beta4, either lacking the cytoplasmic COOH-terminal half or carrying phenylalanine substitutions in the tyrosine activation motif of the cytoplasmic domain, the recombinant BP180 molecules were mostly not colocalized with alpha6beta4, but remained diffusely distributed at the cell surface. Moreover, in cells transfected with cDNAs for alpha6A and a beta4/beta1 chimera, in which the cytoplasmic domain of beta4 was replaced by that of the beta1 integrin subunit, BP180 was not colocalized with the alpha6beta4/beta1 chimera in focal adhesions, but remained again diffusely distributed. These results indicate that sequences within the cytoplasmic domain of beta4 determine the subcellular distribution of BP180.

Figures

Figure 1
Figure 1
(A) Schematic representation of wild-type and mutant forms of BP180 with summary of their localization in 804G cells. The upper three cDNA-constructs represent wild-type (clone A) and COOH-terminal truncations of BP180 (clones B and C), while the lower five (clones D, E, F, G, and H) represent chimeric cDNA-constructs encoding the membrane localization sequence of K-Ras fused with cDNAs encoding various cytoplasmic regions of BP180. IC, intracellular domain; EC, extracellular domain; T, FLAG™ tag; DSR, degenerate set of four 24-26 residue tandem repeats; TM, membrane-spanning domain; CX, membrane localization sequences of K-Ras. Truncations introduced by cloning procedures are indicated by the segment of amino acids that were deleted (Δ). The protein sequence of BP180 is numbered according to Hopkinson et al. (18). (B) Schematic representation of α6A and β4A integrin subunits, of mutant form β4A1382, and of the β4/β1 chimera. The mutant form β4A1382 lacks the COOH-terminal half of the cytoplasmic domain, including the third and fourth type III fibronectin repeats (FNIII) and a portion of the connecting segment (CS). The β4/β1 chimera consists of the extracellular and the transmembrane domain of β4 fused to the entire cytoplasmic domain of β1. The position of the two tyrosine residues, Tyr1422 and Tyr1440, in the cytoplasmic domain of β4, which are part of the tyrosine activation motif, is indicated (arrows). The COOH-terminal truncation (Δ) is indicated by the stretch of amino acids that was removed. The protein sequence of β4A is numbered according to Suzuki and Naitoh (51).
Figure 2
Figure 2
Identification of mutant forms of BP180 expressed in 804G cells by immunoblotting. Extracts of cells transfected with clones A (lanes 1 and 3), B (lane 7), C (lane 8), D (lanes 4 and 9), H (lane 10), E (lane 11), F (lane 12) or G (lanes 13 and 15) and of mock-transfected controls (lanes 2, 5, 14, and 16–18) were processed using the BP180 antiserum J17 (lanes 1 and 2) and the mAb 1A8c (lanes 3–5, and 7 to 14), that are both directed against the intracellular portion of human BP180, as well as the mAb antiFLAG™M2 against the FLAG™ tag (lanes 15 and 16). Samples were separated by 8.5% (lanes 1–6) and 13% (lanes 7–18) SDS-PAGE under reducing conditions. Note that the rabbit antiserum J17 clearly recognizes a protein corresponding to endogenous wild-type BP180 (lanes 1 and 2) with a slightly slower electrophoretic mobility than full-length human BP180 (lane 1). The mutant protein encoded by clone G, which carried the largest deletion of the cytoplasmic domain, is not recognized by mAb 1A8c (lane 13), but is recognized by the mAb anti-FLAG™M2 (lane 15) (arrow head). Since mAb anti-FLAG generated high unspecific background (lanes 15 and 16), only short time exposure is depicted. Mock-transfected cells were also processed using an antiserum against the cytoplasmic domain of β4 (lane 6), an anti-α6A antiserum recognizing the light chain of the endogenous α6 (lane 17), and a normal rabbit serum (lane 18). Molecular weight markers are indicated in kD.
Figure 3
Figure 3
Immunolocalization of mutant forms of BP180 in hemidesmosomes of transfected 804G cells by confocal laser microscopy. Cells were grown on glass coverslips and transfected with clones A (A and B), B (C and D) or C (E and F). After 36 h, cells were fixed with 1% formaldehyde, permeabilized with 0.5% Triton X-100, and subjected to double immunofluorescence using the mAb anti-FLAG™M2 (A, C, and E) and an anti-α6A antiserum (B, D, and F). FITC-conjugated goat anti–mouse IgG (left) and Texas red–conjugated donkey anti–rabbit IgG (right). The recombinant forms of BP180 were concentrated in a Swiss cheese–like pattern characteristic for hemidesmosome-like structures, where they were colocalized with α6 along the basal cell surface as demonstrated by z-sections of individual cells (insets). Note that the mutant form expressed by clone C showed, in addition to its localization in hemidesmosomes, a diffuse cytoplasmic distribution (E). Bar, 10 μm.
Figure 4
Figure 4
Immunolocalization of chimeric forms of BP180 composed of the membrane targeting sequence of K-Ras combined with various cytoplasmic portions of BP180 in 804G cells. Cells transfected with clones D (A and B), E (C and D), F (E and F), G (G and H), or H (I and J) were fixed, permeabilized, and subjected to double immunofluorescence using the mAb antiFLAG™M2 (A, C, E, G, and I) and an anti-α6A antiserum (B, D, F, H, and J). FITC-conjugated goat anti–mouse IgG (left) and Texas red–conjugated donkey anti–rabbit IgG (right). In cells transfected with clones D, E, or F, the chimeric proteins are clearly codistributed with α6 and are concentrated along the basal cell surface (z-sections in the insets). In contrast, the chimeric proteins expressed by clone G (G) or H (I) are diffusely distributed at the cell surface and are not colocalized with α6. Bar, 10 μm.
Figure 5
Figure 5
Immunolocalization of clone B encoded protein, in which combined alanine substitutions of three serine residues (Ser169, Ser175, and Ser180) were introduced. 804G cells were grown on glass coverslips, transfected, fixed with 1% formaldehyde, permeabilized with 0.5% Triton X-100, and subjected to double immunofluorescence using the mAb anti-FLAG™M2 (A) and an anti-α6A antiserum (B). FITC-conjugated goat anti–mouse IgG (left) and Texas red–conjugated donkey anti– rabbit IgG (right). The recombinant form of BP180 was distributed in a Swiss cheese–like pattern characteristic for hemidesmosome-like structures, where it was colocalized with α6. Bar, 10 μm.
Figure 9
Figure 9
(A) Immunoprecipitation analysis of transfected COS-7 cells. Lysates of 35S-radiolabeled cells cotransfected with cDNAs for α6A and β4A as well as clone D (lanes 1–3), clone B (lanes 4–6), or clone A (lanes 7–9), with cDNAs for α6A and β4A alone (lanes 10–12), and of mock-transfected cells (lanes 13–15), were immunoprecipitated with the mAb anti-FLAG™M2 (lanes 1, 4, 7, 10, and 13), an anti-α6 antiserum (lanes 2, 5, 8, 11, and 14), and an anti-β4 antiserum (lanes 3, 6, 9, 12, and 15). Note that immunoprecipitation of cells transfected with cDNAs for α6A and β4A with either an anti-α6 and an anti-β4A antiserum yielded a heterodimer complex of α6A and β4A, while no coprecipitation of BP180 is observed. No coprecipitation of the α6A and β4A is detected by using the mAb anti-FLAG™M2, even after longer exposure of the gel. (B) Lysates of 35S-radiolabeled cells cotransfected with cDNA for α6A as well as with clone D (lanes 1–3), clone B (lanes 4–6), or clone A (lanes 7– 9), and of cells cotransfected with cDNA for β4A and clone D (lanes 10–12), clone B (lanes 13–15), or clone A (lanes 16–18). Immunoprecipitation was performed with the mAb anti-FLAG™M2 (lanes 1, 4, 7, 10, 13, and 16), an anti-α6 antiserum (lanes 2, 5, 8, 11, 14, and 17), and an anti-β4 antiserum (lanes 3, 6, 9, 12, 15, and 18). No coprecipitation of the α6A and β4A is found by using the mAb antiFLAG™M2. In addition, no coprecipitation of mutant BP180 proteins is detected with either an anti-α6 or an anti-β4A antiserum. Note that one radiolabeled polypeptide (arrow heads) is coprecipitated by the mAb anti-FLAG™M2 from extracts of cells transfected with clone B. The identity of this protein, which was not precipitated when cells were lysed with 1% Triton X-100 (not shown), is unclear. Samples were analyzed by 8% SDS-PAGE under reducing conditions. Molecular mass markers are indicated in kD.
Figure 6
Figure 6
Confocal double immunofluorescence microscopy of transfected COS-7 cells showing the subcellular distribution of the chimeric protein composed of the membrane targeting sequence of K-Ras combined with the cytoplasmic domain of BP180 (clone D). Cells grown on glass coverslips were transfected with clone D. After 36 h, cells were fixed, permeabilized, and subjected to double labeling using the BP180 antiserum J17 (A and C) and the mAb VIN 11-5 against vinculin (B), or the mAb 121 against HD1 (D). Texas red–conjugated donkey anti–rabbit IgG (left) and FITC-conjugated goat anti– mouse IgG (right). The chimeric BP180 protein is distributed diffusely at the cell surface and is not colocalized with endogenous vinculin or HD1. Bar, 10 μm.
Figure 7
Figure 7
Double immunofluorescence microscopy of transfected COS-7 showing that the chimeric protein composed of the membrane targeting sequence of K-Ras combined with the entire cytoplasmic domain of BP180 (clone D) is codistributed with the α6β4 integrin. Cells grown on glass coverslips were transfected with clone D (A–F) together with cDNAs for human α6A and β4A (A–D), or with cDNAs for human α6A and β4A1382 (E and F). After 36 h cells were fixed, permeabilized, and subjected to double labeling using the mAb antiFLAG™M2 (A and E) and the rat mAb GoH3 (B and F) as well as the BP180 antiserum J17 (C) and the mAb 121 against HD1(D). FITCconjugated goat anti–mouse IgG (A, D, and E), Texas red–conjugated donkey anti– rabbit IgG (C). The rat mAb GoH3 was stained using a rabbit anti–rat IgG (absorbed against mouse IgG) and Texas red–conjugated donkey anti–rabbit IgG (B and F). In cells transfected with α6A and β4A cDNAs, the BP180 chimeric protein is found concentrated along the basal cell surface, where it is codistributed with α6 and HD1. In contrast, in cells cotransfected with cDNAs for α6A and β4A1382, the mutant form of BP180 displayed uniquely an apicolateral cell surface distribution and is not colocalized with α6. Z-sections of transfected cells are shown in the insets. The optical plane is the same as in Fig. 6. Bar, 10 μm.
Figure 8
Figure 8
Double immunofluorescence microscopy of transfected COS-7 showing that the subcellular distribution of clone B encoded protein is affected by the cytoplasmic domain of the β4 integrin subunit. Cells were transfected with clone B (A–F) together with cDNAs for human α6A and β4A (A and B), or with cDNAs for human α6A and a mutated β4 carrying phenylalanine substitutions of the TAM (C and D), or, finally, with cDNAs for α6A and a β4/β1 chimera, in which the cytoplasmic domain of β4 was replaced by that of β1 (E and F). After 36 h cells were fixed, permeabilized, and subjected to double labeling using the mAb anti-FLAG™M2 (A, C, and E) and the rat mAb GoH3 (B, D, and F). FITC-conjugated goat anti–mouse IgG (A, C, and E). The rat mAb GoH3 was stained using a rabbit anti–rat IgG (absorbed against mouse IgG) and Texas red–conjugated donkey anti–rabbit IgG (B, D, and F). In cells transfected with α6A and β4A cDNAs, the clone B encoded protein was localized along the basal cell surface codistributing with α6β4. In contrast, in cells cotransfected with cDNAs for α6A and a β4 carrying a mutated TAM or a β4/β1 chimera, the BP180 recombinant molecule displayed predominantly an apicolateral cell surface distribution and is not colocalized with α6β4 or α6β4/β1, respectively. Z-sections of transfected cells are shown in the insets. The optical plane is the same as in Fig. 6. Bar, 10 μm.

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