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

The α6β4 laminin binding integrin functions in the assembly of type I hemidesmosomes, which are specialized cell-matrix adhesion sites found in stratified epithelial cells. Although endothelial cells do not express all the components of type I hemidesmosomes, endothelial cells can express the α6β4 integrin. Because endothelial cells lose expression of α6β4 in culture, we expressed recombinant α6β4 in the dermal microvascular endothelial cell line, HMEC-1, to test whether endothelial cells can assemble adhesion structures containing α6β4. Using immunofluorescence microscopy, we found that recombinant α6β4 concentrates specifically in a novel fibrillar structure on the basal surface of endothelial cells in the absence of an exogenous laminin substrate. This localization is regulated by an intracellular mechanism, because the β4 cytoplasmic domain is sufficient to direct a reporter domain (IL-2R) to the fibrillar structures independently of recombinant α6β4. In addition, this IL2R-β4 chimera is sufficient to recruit the intermediate filament-associated protein HD1/plectin to these fibrillar structures and this also occurs in the absence of recombinant α6β4. The fibrillar localization pattern, as well as the recruitment of HD1/plectin, requires the first and second fibronectin type III repeats and the connecting segment of the β4 tail. In addition, when endothelial cells are provided a laminin 5-rich matrix, recombinant α6β4 redistributes from the fibrillar structure to type I hemidesmosome-like structures. The β4 cytoplasmic domain can also direct a reporter domain to these type I hemidesmosome-like structures; however, this process is dependent upon the expression of recombinant α6β4. Biochemical analysis indicates that both the fibrillar and the type I hemidesmosome-like structures are associated with the vimentin intermediate filament cytoskeleton. Thus, the results illustrate that endothelial cells have the essential components necessary to assemble at least two distinct α6β4-containing and vimentin-associated structures on their basal surface and that the β4 cytoplasmic tail and the availability of specific α6β4 ligands regulate receptor localization to these structures.