CATENIN PROTEINS MEDIATING CADHERIN AND CELL FUNCTIONS

钙粘附素细胞-细胞黏附蛋白在这两个过程中发挥核心作用 建立(形态发生)和维持(肿瘤抑制)， 细胞的分化状态。钙粘附素存在于所有脊椎动物固体中 组织，可能也是无脊椎动物组织常见的。高度的 钙粘附素的保守胞质结构域与三个相关(到目前为止) 鉴定出的多肽称为α-连环素、β-连环素和白蛋白 (γ-连环蛋白)，它们是钙粘附素介导的细胞-细胞所必需的 粘附力。最近还发现了α-连环蛋白和β-连环蛋白 与细胞内抑癌基因产物APC相关，因此 增加了连环蛋白已知的相互作用和潜力的复杂性 功能。β-连环蛋白和白蛋白是两种不同的蛋白质 相同的细胞，但在结构和结构上高度同源 氨基酸序列。它们的特定结构与共享结构-功能有 尚未确定，但连环蛋白被认为是共同的 调节钙粘附素(和APC？与肌动蛋白细胞骨架的相互作用 -很可能与我们希望识别的其他细胞内蛋白一起。 我们最近还获得了证据表明，β-连环蛋白 参与Wnt发育信号通路。 我们的研究以三个具体目标为指导： A)确定β-连环蛋白的结构-功能。 B)确定β-连环蛋白的结构-功能。 C)发现并鉴定其他相互作用的细胞质蛋白质 含钙粘附素或连环蛋白。 为达到这些目标，所采用的试验系统将包括 非洲爪哇卵和胚胎、犬MDCK和非洲爪哇A6细胞培养 品系和酵母“双杂交”系统。表位标记的突变构建体 β-连环蛋白和白蛋白，以及β-连环蛋白-白蛋白嵌合体 将其导入MDCK和A6组织培养细胞，并 所产生的表型使用相位对比和 免疫荧光显微镜。突变的白蛋白结构也将是 在非洲爪哇胚胎中进行测试，以检查其结构-功能(相对于 β-连环蛋白)在发育背景下。免疫沉淀法 表位标记的β-连环蛋白和白蛋白构建物将允许 导致观察到的细胞内相互作用的鉴定 表型。与钙粘附素-连环蛋白复合体相互作用的新蛋白质 将通过化学交联剂和酵母双杂交进行鉴定 接近了。

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.