A minimal region on the integrin beta4 subunit that is critical to its localization in hemidesmosomes regulates the distribution of HD1/plectin in COS-7 cells.

The integrin alpha6 beta4 is a major component of hemidesmosomes, in which it mediates firm adhesion to laminin 5. Previous studies have shown that the incorporation of alpha6 beta4 into hemidesmosomes requires a 303 amino acid stretch of the cytoplasmic domain of beta4, comprising part of the first fibronectin type III (FNIII) repeat, the second FNIII repeat and the segment that connects the second to the third FNIII repeat (connecting segment). Now, we have further defined sequences within beta4 that are critical for its localization in hemidesmosomes and we demonstrate that these sequences also induce the redistribution of HD1/plectin into junctional complexes containing the integrin alpha6 beta4 in COS-7 cells, transfected with cDNAs encoding alpha6A and beta4. Truncation of the cytoplasmic domain of beta4 after amino acids 1,382 or 1,355 in the connecting segment, by which a potential tyrosine activation motif (TAM) is removed, does not prevent the localization of alpha6 beta4 in hemidesmosomes in the rat bladder carcinoma cell line 804G and neither did it eliminate the ability of alpha6 beta4 to change the subcellular distribution of HD1/plectin in COS-7 cells. In contrast, beta4 subunits in which the entire connecting segment had been deleted or which were truncated after amino acid 1,328, which removes almost the complete segment, had lost both of these functions. Furthermore, when beta4 subunits with either a deletion of the second FNIII repeat or a small deletion in this repeat were co-expressed with alpha6, the integrins were not localized in hemidesmosomes and did not induce the redistribution of HD1/plectin in COS-7 cells. Finally, the fourth FNIII repeat of beta4 could not replace the second in either of these activities. These findings establish that a region in beta4, which encompasses the second FNIII repeat and a stretch of 27 amino acids (1,329-1,355) of the connecting segment, is critical for the localization of alpha6beta4 in hemidesmosomes and that it regulates the distribution of HD1/plectin.

Integrin alpha 6 beta 4 forms a complex with the cytoskeletal protein HD1 and induces its redistribution in transfected COS-7 cells.

The integrin alpha 6 beta 4 is a major component of hemidesmosomes, in which it is linked to intermediate filaments. Its presence in these structures is dependent on the beta 4 cytoplasmic domain but it is not known whether beta 4 interacts directly with keratin filaments or by interaction with other proteins. In this study, we have investigated the interaction of GST-cyto beta 4A fusion proteins with cellular proteins and demonstrate that a fragment of beta 4A, consisting of the two pairs of fibronectin type III repeats, separated by the connecting segment, forms a specific complex containing a 500-kDa protein that comigrates with HD1, a hemidesmosomal plaque protein. A similar protein was also bound by a glutathione S-transferase fusion protein containing the cytoplasmic domain of a variant beta 4 subunit (beta 4B), in which a stretch of 53 amino acids is inserted in the connecting segment. Subsequent immunoblot analysis revealed that the 500-kDa protein is in fact HD1. In COS-7 cells, which do not express alpha 6 beta 4 or the hemidesmosomal components BP230 and BP180, HD1 is associated with the cytoskeleton, but after transfecting the cells with cDNAs for human alpha 6 and beta 4, it was, instead, colocalized with alpha 6 beta 4 at the basal side of the cells. The organization of the vimentin, keratin, actin, and tubulin cytoskeletal networks was not affected by the expression of alpha 6 beta 4 in COS-7 cells. The localization of HD1 at the basal side of the cells depends on the same region of beta 4 that forms a complex containing HD1 in vitro, since the expression of alpha 6 with a mutant beta 4 subunit that lacks the four fibronectin type III repeats and the connecting segment did not alter the distribution of HD1. The results indicate that for association of alpha 6 beta 4 with HD1, the cytoplasmic domain of beta 4 is essential. We suggest that this association may be crucial for hemidesmosome assembly.

The subcellular distribution of the high molecular mass protein, HD1, is determined by the cytoplasmic domain of the integrin beta 4 subunit.

The high molecular mass protein, HD1, is a structural protein present in hemidesmosomes as well as in distinct adhesion structures termed type II hemidesmosomes. We have studied the distribution and expression of HD1 in the GD25 cells, derived from murine embryonal stem cells deficient for the beta 1 integrin subunit. We report here that these cells possess HD1 but not BP230 or BP180; two other hemidesmosomal constituents, and express only traces of the alpha 6 beta 4 integrin. By immunofluorescence and interference reflection microscopy HD1 was found together with vinculin at the end of actin filaments in focal contacts. In OVCAR-4 cells, derived from a human ovarian carcinoma which, like GD25 cells, only weakly express alpha 6 beta 4, HD1 was also localized in focal contacts. Upon transfection of both GD25 and OVCAR-4 cells with cDNA for the human beta 4 subunit the subcellular distribution of HD1 changed significantly. HD1 is then no longer present in focal contacts but in other structures at cell-substrate contacts, colocalized with alpha 6 beta 4. These junctional complexes are probably the equivalent of the type II hemidesmosomes. Transfection of GD25 cells with beta 1 cDNA did not affect the distribution of HD1, which indicates that the localization of HD1 in focal contacts was not due to the absence of beta 1. Moreover, in GD25 cells transfected with cDNA encoding a beta 4/beta 1 chimera, in which the cytoplasmic domain of beta 4 was replaced by that of beta 1, the distribution of HD1 was unaffected. Our findings indicate that the cytoplasmic domain of beta 4 determines the subcellular distribution of HD1 and emphasize the important role of alpha 6 beta 4 in the assembly of hemidesmosomes and other junctional adhesive complexes containing HD1.

Deficiency of the integrin beta 4 subunit in junctional epidermolysis bullosa with pyloric atresia: consequences for hemidesmosome formation and adhesion properties.

Junctional epidermolysis bullosa (JEB) comprises a group of inherited autosomal recessive blistering disorders characterized by dermo-epidermal separation through the lamina lucida of the basement membrane. We identified a patient with JEB associated with pyloric atresia (PA), in whom the integrin beta 4 subunit was completely absent. At the ultrastructural level, the hemidesmosomes were reduced in number, appeared rudimentary and lacked a subbasal dense plate and frequently an inner attachment plaque. However, keratin filaments were still anchored to the cytoplasmic plaque of the hemidesmosome. Immunofluorescence analysis showed that the beta 4 subunit was absent in the skin of the PA-JEB patient, whereas the alpha 6 subunit appeared to be normally distributed along the basement membrane zone, as were the other hemidesmosomal components BP230, BP180 and HD1. Furthermore, the alpha 3 and beta 1 subunits were not only detected at the lateral membranes of basal cells in PA-JEB skin, as in normal skin, but also along the basement membrane zone. The few hemidesmosome-like structures found in cultured keratinocytes from the PA-JEB patient contained the hemidesmosomal components BP230, BP180 and HD1, but not the integrin alpha 6 subunit. Like alpha 3, this subunit was colocalized with vinculin in focal contacts at the ends of actin stress fibers. Immunoprecipitation analysis revealed that alpha 6 was associated with beta 1 on PA-JEB keratinocytes, whereas normal human keratinocytes (NHKs) exclusively express alpha 6 beta 4 on their cell surface. The initial adhesion of PA-JEB and normal keratinocytes to laminin-1 and laminin-5, both ligands for alpha 6 beta 1 and alpha 6 beta 4, was similar. In migration assays, the PA-JEB keratinocytes were more motile on laminin-5 than normal keratinocytes. Our observations indicate that the integrin alpha 6 beta 4 plays a crucial role in the proper assembly of hemidesmosomes and in the stabilization of the dermal-epidermal junction. The fragility of the skin and the blistering in this patient appear to have been due to the deficiency of the integrin beta 4 subunit, which results in the formation of too few and structurally abnormal hemidesmosomes.

The alpha 6 beta 4 integrin is a receptor for both laminin and kalinin.

Previously, we have establish K562 transfectants that express either alpha 6A beta 1 or alpha 6B beta 1 (K alpha 6A or K alpha 6B) on their surface. Both cell lines bind to laminin and kalinin after treatment with the beta 1-stimulatory antibody TS2/16. Here we introduce the full-length beta 4 cDNA into the alpha 6A- and alpha 6B-expressing K562 cells and selected stably transfected cells. The beta 4 subunit was expressed on the surface of both transfectants and it formed dimers with the alpha 6A or alpha 6B subunits. Immunoprecipitation and preclearing analyses revealed that both transfectants expressed alpha 6 beta 1, in addition to alpha 6 beta 4. While K alpha 6A and K alpha 6B cells required TS2/16 stimulation for binding to laminin or kalinin, adhesion of the unstimulated beta 4-transfected K alpha 6A and K alpha 6B cells to these matrix components was already substantial. This adhesion was mediated by both alpha 6 beta 1 and alpha 6 beta 4 since it was completely blocked by an alpha 6-specific antibody or by a combination of anti-beta 1 and anti-beta 4 antibodies, but only partially by either of these latter two antibodies alone. Adhesion to laminin was completely blocked by an antiserum to laminin fragment E8 as was the adhesion to kalinin by an antibody to kalinin, demonstrating the specificity of adhesion. Both transfectants always adhered more strongly to kalinin than to laminin. Furthermore, binding to kalinin was less well blocked by antibodies to beta 4 than binding to laminin, indicating that the affinity of alpha 6 beta 4 for kalinin is higher than that for laminin. The fact that alpha 6 beta 1 mediated adhesion without TS2/16 stimulation on the beta 4-transfected K alpha 6A and K alpha 6B cells suggests that some activation of alpha 6 beta 1 had occurred in these cells, even though binding was increased when they were actively stimulated by the antibody TS2/16. Finally, we show that Mn2+ induced binding of solubilized alpha 6 beta 4 to matrix containing kalinin, deposited by the murine cell line RAC-11P/SD. This binding was inhibited by the anti-alpha 6 mAb GoH3. Together, these results indicate that both alpha 6 beta 1 and alpha 6 beta 4 are receptors for laminin and kalinin and that there are no differences in ligand specificity between the A and B variants of the alpha 6 subunit when associated with either beta 1 or beta 4.