HEMIDESMOSOMAL PROTEIN LINKAGE MAP BY TWO HYBRID SYSTEM

研究人员建议使用酵母双杂交方法与 BPAG2的胞外区、胞外区和胞内区 A6和b4整合素结构域作为筛选cDNA文库的“诱饵” 相互作用的多肽。假定的交互作用将使用以下方法进行确认 免疫共沉淀分析和结合分析。一旦确认，cDNA3 克隆将进一步表征其基因组位置 确定；将产生抗体，Northern印迹分析，在 将使用原位杂交和免疫细胞化学来表征 这些蛋白质的表达。 半桥粒是上皮基底板的主要部位。 附着；半染色体结构/功能缺陷导致 人类中的水泡性疾病。一些半染色体组分 已被鉴定，其中包括完整的膜蛋白 BPAG2/BP180(具有胶原样胞外域)和 整合素对a6b4(与层粘连蛋白5相互作用)。胞外 这些蛋白质的结构域与含有vii的胶原蛋白相互作用。 锚定纤维，将半桥粒锚定在基板上。在……里面 细胞质BPAG1e、plectin、p2OO和IFAP300已被提出 介导角蛋白类中间丝对血管的锚定作用 半桥粒。虽然这些蛋白质之间的一些相互作用 已经确定，它们的全部交互作用仍有待于 被定义。也很可能有更多的半染色体 蛋白质仍有待鉴定。酵母双杂交系统是 因此，一个合理的方法来鉴定新的多肽和 研究它们之间的相互作用。作者的初步意见 结果表明，他们已经掌握了这方面的技术方面 方法。已经开发的数据表明，一种来自 VII型胶原与凝血酶原蛋白I相互作用(已有相互作用 由“常规”方法定义)。一种来自细胞质的诱饵 BPAG2/BP180的结构域提示与细胞质结构域相互作用 在b4整合素中，plectin的C-末端结构域，N-末端结构域 在src底物和Aradillo-Repeat蛋白p120的C末端 角蛋白18的结构域和一种与桥粒蛋白相关的新多肽 和envoplakin(除了其他新的假定的相互作用因子)。

The investigator proposes to use a yeast two-hybrid approach with the extracellular domain of BPAG2, and the extracellular and intracellular domains of a6 and b4 integrins as "bait" to screen cDNAs libraries for interacting polypeptides. Putative interactors will be confirmed using co-immunoprecipitation analysis and binding assays. Once confirmed, cDNA clones will be further characterized and their genomic positions determined; antibodies will be generated, Northern blot analysis, in situ hybridization and immunocytochemistry will be used to characterize the expression of these proteins. The hemidesmosome is the primary site of epithelial-basal lamina attachment; defects in hemidesmosomal structure/function lead to blistering diseases in humans. A number of hemidesmosomal components have been identified, these include the integral membrane proteins BPAG2/BP180 (which has a collagen-like extracellular domain) and the integrin pair a6b4 (which interacts with laminin 5). The extracellular domains of these proteins interact with collagen VII-containing anchoring fibers to anchor the hemidesmosome to the basal lamina. In the cytoplasm BPAG1e, plectin, p2OO and IFAP300 have been proposed to mediate the anchorage of keratin-type intermediate filaments to the hemidesmosome. While some of the interactions between these proteins have been identified, the full range of their interactions remains to be defined. It is also very likely that many more hemidesmosomal proteins remain to be identified. The yeast two-hybrid system is therefore a reasonable approach to identifying new polypeptides and studying their interactions with one another. The author's preliminary results indicate that they have mastered the technical aspects of this method. Data already developed indicate that a bait derived from collagen VII interacts with thrombospondin I (an interaction already defined by 'conventional' methods). A bait derived from the cytoplasmic domain of BPAG2/BP180 suggests interactions with the cytoplasmic domain of b4 integrin, the C-terminal domain of plectin, the N-terminal domain of the src-substrate and armadillo-repeat protein p120, the C-terminal domain of the keratin 18, and a novel polypeptide related to desmoplakin and envoplakin (in addition to other novel putative interactors).