A minimal region on the integrin β4 subunit that is critical to its localization in hemidesmosomes regulates the distribution of HD1/plectin in COS-7 cells

Abstract

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.

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

“…Therefore, the results indicate that the region of the β4 intracellular domain required for its fibrillar localization in endothelial cells is contained within the first pair of Fn type III repeats and the CS. This is the same region of the β 4 intracellular domain determined to be required for the localization of α6β4 to type I hemidesmosomes in 804G rat bladder carcinoma cells and type II hemidesmosome-like structures in fibroblastic cells (Spinardi et al, 1993;Niessen et al, 1997a). This result suggests that similar intracellular protein interactions with the β4 cytoplasmic tail may be involved in regulating receptor localization to all three structures.…”

“…The same fibrillar localization pattern was observed for recombinant α6β4 when expressed in early passage primary dermal microvascular endothelial cells (data not shown). This α6β4-containing structure appears to be distinct from the previously described type II hemidesmosomes in epithelial cells and type II-like hemidesmosomes in fibroblasts (Uematsu et al, 1994;Niessen et al, 1997a). The majority of recombinant α6β4 concentrates in fibrillar structures where vinculin is not detected (data not shown).…”

“…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 motifs include two pairs of FN type III structural repeats separated by a connecting segment (CS), which contains a tyrosineactivation motif that has been shown to function in signal transduction in other receptors (Timson-Gauen et al, 1996). It has previously been shown that a region of the β4 intracellular domain contained within the first pair of FN type III repeats and the CS is involved in regulating the localization of α6β4 to type I hemidesmosomes in rat bladder carcinoma 804G cells and type II hemidesmosome-like structures in fibroblastic cells (Niessen et al, 1997a;Spinardi et al, 1993). To determine whether the same or distinct regions of the β4 cytoplasmic domain are important for mediating the interactions directing the chimera to the fibrillar structures in endothelial cells, we constructed chimeric receptors containing various deletions in the β4 intracellular domain, focusing on the region of the β4 tail containing the FN type III repeats and the CS (Fig.…”

“…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.…”