CATENIN PROTEINS MEDIATING CADHERIN AND CELL FUNCTIONS

钙粘蛋白细胞-细胞粘附蛋白在两种细胞粘附中起着重要作用。 建立（形态发生）和维持（肿瘤抑制）， 细胞的分化状态。 钙粘蛋白存在于所有脊椎动物的固体中， 组织，并且也可能是无脊椎动物组织所共有的。 高度 钙粘蛋白的保守胞质结构域与三个（thus-far） 一种被鉴定为α-连环蛋白、β-连环蛋白和斑珠蛋白的多肽 （γ-连环蛋白），这是必不可少的钙粘蛋白介导的细胞-细胞 粘连 最近还发现了α-连环蛋白和β-连环蛋白 与细胞内肿瘤抑制基因产物APC相关，因此 增加了连环蛋白的已知相互作用和潜力的复杂性， 功能协调发展的 β-连环蛋白和斑珠蛋白是不同的蛋白质， 相同的细胞，但在结构上彼此高度同源， 氨基酸序列 它们的特定结构功能与共享结构功能具有 尚未确定，但连环蛋白集体被认为是 介导钙粘蛋白（和APC的？与肌动蛋白细胞骨架的相互作用 - 并且很可能与我们希望鉴定的其他细胞内蛋白质有关。 我们最近也有证据表明β-连环蛋白 参与Wnt发育信号通路。 我们的研究遵循三个具体目标： A）定义β-连环蛋白的结构-功能。 B）定义β-连环蛋白的结构和功能。 C）发现和表征与细胞质相互作用的其他细胞质蛋白 与钙粘蛋白或连环蛋白结合。 为了达到这些目标，所采用的实验系统将包括 爪蟾卵和胚胎，犬MDCK和爪蟾A6细胞培养物 线，和酵母“双杂交”系统。 表位标记的突变体构建体 β-连环蛋白和斑珠蛋白，以及β-连环蛋白-斑珠蛋白嵌合体 将构建体转染到MDCK和A6组织培养细胞中，并且 使用相差评价所得表型， 免疫荧光显微术。 突变体斑珠蛋白构建体也将是 在非洲爪蟾胚胎中进行测试，以检查其结构功能（相对于 β-连环蛋白）的基因。 的免疫沉淀 表位标记的β-连环蛋白和斑珠蛋白构建体将允许 识别导致观察到的细胞内相互作用 表型 与钙粘蛋白-连环蛋白复合物相互作用的新蛋白质 将采用化学交联剂和酵母双杂交技术进行鉴定 接近。

Cadherin cell-cell adhesion proteins play central roles in both the establishment (morphogenesis), and maintenance (tumor suppression), of a cell's differentiated state. Cadherins are present in all vertebrate solid tissues, and may be common to invertebrate tissues as well. The highly conserved cytoplasmic domains of cadherins associate with three (thus-far) identified polypeptides named alpha-catenin, beta-catenin, and plakoglobin (gamma-catenin), which are essential to cadherin-mediated cell-cell adhesion. alpha-Catenin and beta-catenin have also recently been found associated with the intracellular tumor-suppresser gene product APC, thus increasing the complexity of the catenins' known interactions and potential functions. beta-Catenin and plakoglobin are distinct proteins found within the same cells, but are highly homologous to each other in structure and amino acid sequence. Their specific versus shared structure-functions have not yet been determined, but the catenins collectively are thought to mediate the cadherins' (and APC's? interaction with the actin cytoskeleton - and likely with other intracellular proteins that we wish to identify. We have also recently obtained evidence suggesting that beta-catenin participates in the Wnt developmental signaling pathway. Our research is guided by three Specific Aims: A) Define the structure-function of beta-catenin. B) Define the structure-function of beta-catenin. C) Discover and characterize additional cytoplasmic proteins that interact with cadherins or catenins. In meeting these objectives, the experimental systems employed will include Xenopus eggs and embryos, cultures of canine MDCK and Xenopus A6 cell lines, and the yeast "two-hybrid" system. Epitope-tagged mutant constructs of beta-catenin and plakoglobin, and beta-catenin-plakoglobin chimeric constructs, will be transfected into MDCK and A6 tissue culture cells, and the resulting phenotypes evaluated using phase-contrast and immunofluorescence microscopy. Mutant plakoglobin constructs will also be tested in Xenopus embryos, to examine its structure-function (relative to that of beta-catenin) in a developmental context. Immunoprecipitation of the epitope-tagged beta-catenin and plakoglobin constructs will permit identification of intracellular interactions leading to the observed phenotypes. Novel proteins interacting with the cadherin-catenin complex will be identified by using chemical cross-linker and yeast two-hybrid approaches.