Shaw L M, Mercurio A M
Program in Cell and Developmental Biology, Harvard Medical School, Boston, Massachusetts 02115.
Mol Biol Cell. 1994 Jun;5(6):679-90. doi: 10.1091/mbc.5.6.679.
Several integrin alpha subunits have structural variants that are identical in their extracellular and transmembrane domains but that differ in their cytoplasmic domains. The functional significance of these variants, however, is unknown. In the present study, we examined the possibility that the A and B variants of the alpha 6 beta 1 integrin laminin receptor differ in function. For this purpose, we expressed the alpha 6A and alpha 6B cDNAs, as well as a truncated alpha 6 cDNA (alpha 6-delta CYT) in which the cytoplasmic domain sequence was deleted after the GFFKR pentapeptide, in P388D1 cells, an alpha 6 deficient macrophage cell line. Populations of stable alpha 6A, alpha 6B, and alpha 6-delta CYT transfectants that expressed equivalent levels of cell surface alpha 6 were obtained by fluorescence-activated cell sorter and shown to form heterodimers with endogenous beta 1 subunits. Upon attachment to laminin, the alpha 6A transfectants extended numerous pseudopodia. In contrast, the alpha 6B transfectants remained rounded and extended few processes. The transfectants were also examined for their ability to migrate toward a laminin substratum using Transwell chambers. The alpha 6A transfectants were three- to fourfold more migratory than the alpha 6B transfectants. The alpha 6-delta CYT transfectants did not attach to laminin in normal culture medium, but they did attach in the presence of Mn2+. The alpha 6-delta CYT transfectants migrated to a lesser extent than either the alpha 6A or alpha 6B transfectants in the presence of Mn2+. The alpha 6 transfectants differed significantly in the concentration of substratum bound laminin required for half-maximal adhesion in the presence of Mn2+:alpha 6A (2.1 micrograms/ml), alpha 6B (6.3 micrograms/ml), and alpha 6-delta CYT (8.8 micrograms/ml). Divalent cation titration studies revealed that these transfectants also differed significantly in both the [Ca2+] and [Mn2+] required to obtain half-maximal adhesion to laminin. These data demonstrate that the A and B variants of the alpha 6 cytoplasmic domain can differentially modulate the function of the alpha 6 beta 1 extracellular domain.
几种整合素α亚基具有结构变体，其胞外结构域和跨膜结构域相同，但胞质结构域不同。然而，这些变体的功能意义尚不清楚。在本研究中，我们研究了α6β1整合素层粘连蛋白受体的A和B变体在功能上是否存在差异。为此，我们在α6缺陷的巨噬细胞系P388D1细胞中表达了α6A和α6B cDNA，以及一种截短的α6 cDNA（α6-ΔCYT），其中在GFFKR五肽后删除了胞质结构域序列。通过荧光激活细胞分选仪获得了表达等量细胞表面α6的稳定α6A、α6B和α6-ΔCYT转染子群体，并显示它们与内源性β1亚基形成异二聚体。附着于层粘连蛋白后，α6A转染子伸出许多伪足。相比之下，α6B转染子保持圆形，伸出的突起较少。还使用Transwell小室检测了转染子向层粘连蛋白基质迁移的能力。α6A转染子的迁移能力比α6B转染子高3至4倍。α6-ΔCYT转染子在正常培养基中不附着于层粘连蛋白，但在存在Mn2+的情况下会附着。在存在Mn2+的情况下，α6-ΔCYT转染子的迁移程度低于α6A或α6B转染子。在存在Mn2+的情况下，α6转染子在半最大黏附所需的基质结合层粘连蛋白浓度上有显著差异：α6A（2.1微克/毫升）、α6B（6.3微克/毫升）和α6-ΔCYT（8.8微克/毫升）。二价阳离子滴定研究表明，这些转染子在获得对层粘连蛋白半最大黏附所需的[Ca2+]和[Mn2+]方面也有显著差异。这些数据表明，α6胞质结构域的A和B变体可以不同地调节α6β1胞外结构域的功能。
