The subcellular distribution of the high molecular mass protein, HD1, is determined by the cytoplasmic domain of the integrin β4 subunit

Abstract

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.

Endothelial cells assemble two distinct α6β4-containing vimentin-associated structures: roles for ligand binding and the β4 cytoplasmic tail

“…Our results suggest that the cellular protein involved in directing the localization of α6β4 and the IL-2R-β4 chimera to these structures is not HD1/plectin, because HD1/plectin cannot be detected in any distinct staining pattern until the wild-type β4 chimera or chimeric mutant β4-∆1488-1752 is expressed. This finding is in agreement with others whose data indicate that the cytoplasmic domain of β4 can directly interact with plectin and can determine the subcellular distribution of HD1/plectin (Sanchez-Aparicio et al, 1997;Rezniczek et al, 1998;Niessen et al, 1997b). However, we and others have not been able to identify a separate region of the β4 intracellular domain involved in regulating receptor localization to the basal cell surface from a region involved in targeting HD1/plectin to these structures.…”

“…The extracellular matrix protein associated with these structures remains unidentified. Interestingly, similar structures can be identified in fibroblastic cells expressing recombinant α 6β4 (Sanchez-Aparicio et al, 1997;Niessen et al, 1997a).…”

“…These structures have been described as α6β4 concentrated in linear streaks flanking focal adhesions containing vinculin (Uematsu et al, 1994). When recombinant α6β4 is expressed in fibroblasts, it also concentrates at sites resembling type II hemidesmosomes (Sanchez-Aparicio et al, 1997;Niessen et al, 1997a). For these reasons, it is possible that the localization of α6β4 in fibroblasts might also be regulated by an intracellular mechanism.…”