Wahl James K, Kim Young J, Cullen Janet M, Johnson Keith R, Wheelock Margaret J
University of Nebraska Medical Center, College of Dentistry and Eppley Cancer Center, Omaha, Nebraska 68198-7696, USA.
J Biol Chem. 2003 May 9;278(19):17269-76. doi: 10.1074/jbc.M211452200. Epub 2003 Feb 25.
Cadherins are calcium-dependent glycoproteins that function as cell-cell adhesion molecules and are linked to the actin cytoskeleton via catenins. Newly synthesized cadherins contain a prosequence that must be proteolytically removed to generate a functional adhesion molecule. The goal of this study was to examine the proteolytic processing of N-cadherin and the assembly of the cadherin-catenin complex in cells that express endogenous N-cadherin. A monoclonal antibody specific for the proregion of human N-cadherin was generated and used to examine N-cadherin processing. Our data show that newly synthesized proN-cadherin is phosphorylated and proteolytically processed prior to transport to the plasma membrane. In addition, we show that beta-catenin and plakoglobin associate only with phosphorylated proN-cadherin, whereas p120(ctn) can associate with both phosphorylated and non-phosphorylated proN-cadherin. Immunoprecipitations using anti-proN-cadherin showed that cadherin-catenin complexes are assembled prior to localization at the plasma membrane. These data suggest that a core N-cadherin-catenin complex assembles in the endoplasmic reticulum or Golgi compartment and is transported to the plasma membrane where linkage to the actin cytoskeleton can be established.
钙黏着蛋白是钙依赖性糖蛋白,作为细胞间黏附分子发挥作用,并通过连环蛋白与肌动蛋白细胞骨架相连。新合成的钙黏着蛋白含有一个前序列,必须通过蛋白水解作用将其去除才能产生功能性黏附分子。本研究的目的是检测N-钙黏着蛋白的蛋白水解加工过程以及在表达内源性N-钙黏着蛋白的细胞中钙黏着蛋白-连环蛋白复合物的组装情况。制备了一种针对人N-钙黏着蛋白前区的单克隆抗体,并用于检测N-钙黏着蛋白的加工过程。我们的数据表明,新合成的前体N-钙黏着蛋白在转运到质膜之前会发生磷酸化和蛋白水解加工。此外,我们还表明,β-连环蛋白和桥粒斑珠蛋白仅与磷酸化的前体N-钙黏着蛋白结合,而p120(ctn)可以与磷酸化和未磷酸化的前体N-钙黏着蛋白都结合。使用抗前体N-钙黏着蛋白的免疫沉淀显示,钙黏着蛋白-连环蛋白复合物在定位到质膜之前就已组装。这些数据表明,核心的N-钙黏着蛋白-连环蛋白复合物在内质网或高尔基体区室中组装,并被转运到质膜,在那里可以与肌动蛋白细胞骨架建立连接。