Sánchez-Aparicio P, Martínez de Velasco A M, Niessen C M, Borradori L, Kuikman I, Hulsman E H, Fässler R, Owaribe K, Sonnenberg A
Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
J Cell Sci. 1997 Jan;110 ( Pt 2):169-78. doi: 10.1242/jcs.110.2.169.
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.
高分子量蛋白HD1是一种结构蛋白，存在于半桥粒以及一种名为II型半桥粒的独特黏附结构中。我们研究了HD1在源自缺乏β1整合素亚基的小鼠胚胎干细胞的GD25细胞中的分布和表达情况。我们在此报告，这些细胞含有HD1，但不含有BP230或BP180（另外两种半桥粒成分），并且仅表达痕量的α6β4整合素。通过免疫荧光和干涉反射显微镜观察发现，HD1与纽蛋白一起存在于黏着斑中肌动蛋白丝的末端。在源自人卵巢癌的OVCAR-4细胞中，与GD25细胞一样，仅微弱表达α6β4，HD1也定位于黏着斑中。用人类β4亚基的cDNA转染GD25和OVCAR-4细胞后，HD1的亚细胞分布发生了显著变化。此时HD1不再存在于黏着斑中，而是存在于细胞与基质接触处的其他结构中，与α6β4共定位。这些连接复合体可能等同于II型半桥粒。用β1 cDNA转染GD25细胞不会影响HD1的分布，这表明HD1在黏着斑中的定位不是由于缺乏β1所致。此外，在转染了编码β4/β1嵌合体（其中β4的胞质结构域被β1的胞质结构域取代）的cDNA的GD25细胞中，HD1的分布不受影响。我们的研究结果表明，β4的胞质结构域决定了HD1的亚细胞分布，并强调了α6β4在半桥粒和其他含有HD1的连接黏附复合体组装中的重要作用。
