Homan S M, Mercurio A M, LaFlamme S E
Department of Physiology and Cell Biology, Albany Medical College, Albany, NY 12208, USA.
J Cell Sci. 1998 Sep;111 ( Pt 18):2717-28. doi: 10.1242/jcs.111.18.2717.
The alpha6beta4 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 alpha6beta4 integrin. Because endothelial cells lose expression of alpha6beta4 in culture, we expressed recombinant alpha6beta4 in the dermal microvascular endothelial cell line, HMEC-1, to test whether endothelial cells can assemble adhesion structures containing alpha6beta4. Using immunofluorescence microscopy, we found that recombinant alpha6beta4 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 beta4 cytoplasmic domain is sufficient to direct a reporter domain (IL-2R) to the fibrillar structures independently of recombinant alpha6beta4. In addition, this IL-2R-beta4 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 alpha6beta4. 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 beta4 tail. In addition, when endothelial cells are provided a laminin 5-rich matrix, recombinant alpha6beta4 redistributes from the fibrillar structure to type I hemidesmosome-like structures. The beta4 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 alpha6beta4 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 alpha6beta4-containing and vimentin-associated structures on their basal surface and that the alpha6beta4 cytoplasmic tail and the availability of specific alph6beta4 ligands regulate receptor localization to these structures.
α6β4层粘连蛋白结合整合素在I型半桥粒的组装中发挥作用，I型半桥粒是在复层上皮细胞中发现的特殊细胞-基质粘附位点。虽然内皮细胞不表达I型半桥粒的所有成分，但内皮细胞可以表达α6β4整合素。由于内皮细胞在培养过程中会失去α6β4的表达，我们在真皮微血管内皮细胞系HMEC-1中表达重组α6β4，以测试内皮细胞是否能组装含有α6β4的粘附结构。通过免疫荧光显微镜观察，我们发现，在没有外源性层粘连蛋白底物的情况下，重组α6β4特异性地集中在内皮细胞基底表面的一种新型纤维状结构中。这种定位受细胞内机制调控，因为β4胞质结构域足以将报告结构域（IL-2R）独立于重组α6β4引导至纤维状结构。此外，这种IL-2R-β4嵌合体足以将中间丝相关蛋白HD1/网蛋白募集至这些纤维状结构，且这一过程同样在没有重组α6β4的情况下发生。纤维状定位模式以及HD1/网蛋白的募集需要β4尾部的第一个和第二个纤连蛋白III型重复序列以及连接段。此外，当为内皮细胞提供富含层粘连蛋白5的基质时，重组α6β4会从纤维状结构重新分布至I型半桥粒样结构。β4胞质结构域也能将报告结构域引导至这些I型半桥粒样结构；然而，这一过程依赖于重组α6β4的表达。生化分析表明，纤维状结构和I型半桥粒样结构均与波形蛋白中间丝细胞骨架相关。因此，结果表明，内皮细胞具备在其基底表面组装至少两种不同的含α6β4且与波形蛋白相关结构所需的基本成分，并且α6β4胞质尾部以及特定α6β4配体的可用性调节受体向这些结构的定位。
