Hanakawa Y, Amagai M, Shirakata Y, Sayama K, Hashimoto K
Department of Dermatology, School of Medicine, Ehime University, Ehime, Japan.
J Cell Sci. 2000 May;113 ( Pt 10):1803-11. doi: 10.1242/jcs.113.10.1803.
Desmosomes contain two types of cadherin: desmocollin (Dsc) and desmoglein (Dsg). In this study, we examined the different roles that Dsc and Dsg play in the formation of desmosomes, by using dominant-negative mutants. We constructed recombinant adenoviruses (Ad) containing truncated mutants of E-cadherin, desmocollin 3a, and desmoglein 3 lacking a large part of their extracellular domains (EcaddeltaEC, Dsc3adeltaEC, Dsg3deltaEC), using the Cre-loxP Ad system to circumvent the problem of the toxicity of the mutants to virus-producing cells. When Dsc3adeltaEC Ad-infected HaCaT cells were cultured with high levels of calcium, E-cadherin and beta-catenin, which are marker molecules for the adherens junction, disappeared from the cell-cell contact sites, and cell-cell adhesion was disrupted. This also occurred in the cells infected with EcaddeltaEC Ad. With Dsg3deltaEC Ad infection, keratin insertion at the cell-cell contact sites was inhibited and desmoplakin, a marker of desmosomes, was stained in perinuclear dots while the adherens junctions remained intact. Dsc3adeltaEC Ad inhibited the induction of adherens junctions and the subsequent formation of desmosomes with the calcium shift, while Dsg3deltaEC Ad only inhibited the formation of desmosomes. To further determine whether Dsc3adeltaEC directly affected adherens junctions, mouse fibroblast L cells transfected with E-cadherin (LEC5) were infected with these mutant Ads. Both Dsc3adeltaEC and EcaddeltaEC inhibited the cell-cell adhesion of LEC5 cells, as determined by the cell aggregation assay, while Dsg3deltaEC did not. These results indicate that the dominant negative effects of Dsg3deltaEC were restricted to desmosomes, while those of Dsc3adeltaEC were observed in both desmosomes and adherens junctions. Furthermore, the cytoplasmic domain of Dsc3adeltaEC coprecipitated both plakoglobin and beta-catenin in HaCaT cells. In addition, beta-catenin was found to bind the endogenous Dsc in HaCaT cells. These findings lead us to speculate that Dsc interacts with components of the adherens junctions through beta-catenin, and plays a role in nucleating desmosomes after the adherens junctions have been established.
桥粒胶蛋白（Dsc）和桥粒芯糖蛋白（Dsg）。在本研究中，我们通过使用显性负性突变体，研究了Dsc和Dsg在桥粒形成过程中所起的不同作用。我们构建了含有E-钙黏蛋白、桥粒胶蛋白3a和桥粒芯糖蛋白3的截短突变体的重组腺病毒（Ad），这些突变体缺失了大部分细胞外结构域（EcaddeltaEC、Dsc3adeltaEC、Dsg3deltaEC），利用Cre-loxP腺病毒系统来规避突变体对病毒产生细胞的毒性问题。当用高水平钙培养Dsc3adeltaEC腺病毒感染的HaCaT细胞时，作为黏着连接标记分子的E-钙黏蛋白和β-连环蛋白从细胞-细胞接触位点消失，细胞-细胞黏附被破坏。在用EcaddeltaEC腺病毒感染的细胞中也出现了这种情况。用Dsg3deltaEC腺病毒感染时，细胞-细胞接触位点的角蛋白插入受到抑制，桥粒的标记物桥粒斑蛋白在核周点状区域被染色，而黏着连接保持完整。Dsc3adeltaEC腺病毒抑制了黏着连接的诱导以及随后随着钙转移而形成的桥粒，而Dsg3deltaEC腺病毒仅抑制桥粒的形成。为了进一步确定Dsc3adeltaEC是否直接影响黏着连接，用E-钙黏蛋白转染的小鼠成纤维细胞L细胞（LEC5）被这些突变腺病毒感染。通过细胞聚集试验测定，Dsc3adeltaEC和EcaddeltaEC都抑制了LEC5细胞的细胞-细胞黏附，而Dsg3deltaEC没有。这些结果表明，Dsg3deltaEC的显性负性作用仅限于桥粒，而Dsc3adeltaEC的显性负性作用在桥粒和黏着连接中均有观察到。此外，Dsc3adeltaEC的细胞质结构域在HaCaT细胞中共沉淀了桥粒斑珠蛋白和β-连环蛋白。另外，发现β-连环蛋白在HaCaT细胞中与内源性Dsc结合。这些发现使我们推测Dsc通过β-连环蛋白与黏着连接的成分相互作用，并在黏着连接建立后在桥粒的形成中发挥作用。
