Malmberg Emily K, Pelaseyed Thaher, Petersson Asa C, Seidler Ursula E, De Jonge Hugo, Riordan John R, Hansson Gunnar C
Department of Medical Biochemistry and Cell Biology, Göteborg University, 413 90 Gothenburg, Sweden.
Biochem J. 2008 Mar 1;410(2):283-9. doi: 10.1042/BJ20071068.
The membrane-bound mucins have a heavily O-glycosylated extracellular domain, a single-pass membrane domain and a short cytoplasmic tail. Three of the membrane-bound mucins,MUC3, MUC12 and MUC17, are clustered on chromosome 7 and found in the gastrointestinal tract. These mucins have C-terminal sequences typical of PDZ-domain-binding proteins. To identify PDZ proteins that are able to interact with the mucins,we screened PDZ domain arrays using YFP (yellow fluorescent protein)-tagged proteins. MUC17 exhibited a strong binding to PDZK1 (PDZ domain containing 1), whereas the binding toNHERF1 (Na+/H+-exchanger regulatory factor 1) was weak.Furthermore, we showed weak binding of MUC12 to PDZK1, NHERF1 and NHERF2. GST (glutathione transferase) pull-down experiments confirmed that the C-terminal tail of MUC17 coprecipitates with the scaffold protein PDZK1 as identified byMS. This was mediated through the C-terminal PDZ-interaction site in MUC17, which was capable of binding to three of the four PDZ domains in PDZK1. Immunostaining of wild-type or Pdzk1-/- mouse jejunum with an antiserum against Muc3(17),the mouse orthologue of human MUC17, revealed strong brushborder membrane staining in the wild-type mice compared with an intracellular Muc3(17) staining in the Pdzk1-/- mice. This suggests that Pdzk1 plays a specific role in stabilizing Muc3(17)in the apical membrane of small intestinal enterocytes.
膜结合黏蛋白具有高度O-糖基化的细胞外结构域、单次跨膜结构域和短的细胞质尾巴。三种膜结合黏蛋白，即MUC3、MUC12和MUC17，聚集在7号染色体上，并在胃肠道中发现。这些黏蛋白具有PDZ结构域结合蛋白典型的C末端序列。为了鉴定能够与黏蛋白相互作用的PDZ蛋白，我们使用黄色荧光蛋白（YFP）标记的蛋白筛选了PDZ结构域阵列。MUC17与PDZK1（含1个PDZ结构域）表现出强烈结合，而与NHERF1（钠/氢交换调节因子1）的结合较弱。此外，我们发现MUC12与PDZK1、NHERF1和NHERF2的结合较弱。谷胱甘肽S-转移酶（GST）下拉实验证实，MUC17的C末端尾巴与质谱鉴定的支架蛋白PDZK1共沉淀。这是通过MUC17中的C末端PDZ相互作用位点介导的，该位点能够与PDZK1的四个PDZ结构域中的三个结合。用抗Muc3(17)（人类MUC17的小鼠同源物）的抗血清对野生型或Pdzk1-/-小鼠空肠进行免疫染色，结果显示，与Pdzk1-/-小鼠细胞内的Muc3(17)染色相比，野生型小鼠的刷状缘膜染色较强。这表明Pdzk1在稳定小肠肠细胞顶端膜中的Muc3(17)方面发挥着特定作用。
