CEACAM1, a cell-cell adhesion molecule, directly associates with annexin II in a three-dimensional model of mammary morphogenesis
CEACAM1, a cell-cell adhesion molecule, directly associates with annexin II in a three-dimensional model of mammary morphogenesis
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DOI：
10.1074/jbc.m309115200
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发表时间：
2003-12-12
影响因子：
4.8
通讯作者：
Shively, JE
中科院分区：
文献类型：
--
作者：
Kirshner, J;Schumann, D;Shively, JE
上皮细胞粘附分子CEACAM1（癌胚抗原细胞粘附分子-1）在结肠癌、前列腺癌、乳腺癌和肝癌中下调。CEACAM1-4S是一种具有4个igg样外结构域和一个短细胞质结构域（14个氨基酸）的剪接形式，与膜联蛋白II直接相关，膜联蛋白II是一种脂质筏相关分子，在许多癌症中也下调。采用谷胱甘肽s -转移酶下拉法鉴定膜联蛋白II，其中CEACAM-4S的细胞质结构域与谷胱甘肽s -转移酶融合，融合蛋白与细胞裂解物孵育，分离蛋白通过质谱法测序。这种相互作用首先通过抗CEACAM1和抗膜联蛋白II抗体的互反免疫沉淀得到证实，其次通过共聚焦激光显微镜显示CEACAM1和膜联蛋白II在Matrigel中生长的乳腺上皮细胞中共定位。此外，CEACAM1在质膜上与四聚体AIIt复合物的组分p11共定位，并在分泌囊泡中与膜联蛋白II共定位。CEACAM1-4S胞质结构域的固定化定向肽与牛AIIt直接结合，其与人AIIt的同源性为98%，表面等离子体共振的平均K-D值约为30 nM，表明功能相关的AIIt与CEACAM1-4S胞质结构域直接结合。
The epithelial cell adhesion molecule CEACAM1 ( carcinoembryonic antigen cell adhesion molecule-1) is down-regulated in colon, prostate, breast, and liver cancer. Here we show that CEACAM1-4S, a splice form with four Ig-like ectodomains and a short cytoplasmic domain ( 14 amino acids), directly associates with annexin II, a lipid raft-associated molecule, which is also downregulated in many cancers. Annexin II was identified using a glutathione S-transferase pull-down assay in which the cytoplasmic domain of CEACAM-4S was fused to glutathione S-transferase, the fusion protein was incubated with cell lysates, and isolated proteins were sequenced by mass spectrometry. The interaction was confirmed first by reciprocal immunoprecipitations using anti-CEACAM1 and anti-annexin II antibodies and second by confocal laser microscopy showing co-localization of CEACAM1 with annexin II in mammary epithelial cells grown in Matrigel. In addition, CEACAM1 co-localized with p11, a component of the tetrameric AIIt complex at the plasma membrane, and with annexin II in secretory vesicles. Immobilized, oriented peptides from the cytoplasmic domain of CEACAM1-4S were shown to directly associate with bovine AIIt, which is 98% homologous to human AIIt, with average K-D values of about 30 nM using surface plasmon resonance, demonstrating direct binding of functionally relevant AIIt to the cytoplasmic domain of CEACAM1-4S.