Annexin II binds progastrin and gastrin-like peptides, and mediates growth factor effects of autocrine and exogenous gastrins on colon cancer and intestinal epithelial cells
Annexin II binds progastrin and gastrin-like peptides, and mediates growth factor effects of autocrine and exogenous gastrins on colon cancer and intestinal epithelial cells
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DOI：
10.1038/sj.onc.1209798
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发表时间：
2007-01-01
期刊：
影响因子：
8
通讯作者：
Owlia, A.
中科院分区：
文献类型：
--
作者：
Singh, P.;Wu, H.;Owlia, A.
我们和其他人报道了正常和癌肠细胞上存在新的原胃泌素(PG)/胃泌素受体。我们早前报道过结肠癌细胞细胞膜上存在33 - 36kda的胃泌素结合蛋白。本研究的目的是鉴定33 - 36 kDa波段的蛋白，并分析其功能意义。用碳二亚胺交联剂将放射性标记的胃泌素与胃泌素/PG反应细胞系的膜蛋白交联。将与配体交联的天然膜蛋白用bbb10进行1000倍的增溶和富集，并用表面增强激光解吸/电离时间光质谱法进行分析。利用ProFound搜索引擎对NCBInr数据库中的肽质量进行了研究。通过基质辅助激光解吸/电离飞行时间质谱法对膜联蛋白II （ANX II）进行了鉴定和确认。由于HCT-116细胞表达自分泌PG，在拉下实验中证实PG与ANX II的原位关联。在体外结合实验中证实PG与ANX II直接结合。为了证实这些观察结果在功能上的重要性，我们构建了肠上皮细胞（IEC-18）和人结肠癌细胞系（HCT-116）表达正义和反义（AS） ANX II rna的克隆。AS克隆对外源性（IEC-18）和自分泌（HCT-116） PG的生长反应明显减弱。因此，我们发现膜相关ANX II结合PG/胃泌素，并部分介导肽的生长因子作用。
We and others have reported the presence of novel progastrin (PG)/gastrin receptors on normal and cancerous intestinal cells. We had earlier reported the presence of 33 - 36 kDa gastrin-binding proteins on cellular membranes of colon cancer cells. The goal of the current study was to identify the protein(s) in the 33 - 36 kDa band, and analyse its functional significance. A carbodiimide crosslinker was used for crosslinking radio-labeled gastrins to membrane proteins from gastrin/PG responsive cell lines. Native membrane proteins, crosslinked to the ligand, were solubulized and enriched by > 1000-fold, and analysed by surface-enhanced laser desorption/ionization-time of light-mass spectrometry. The peptide masses were researched against the NCBInr database using the ProFound search engine. Annexin II (ANX II) was identified, and confirmed by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. As HCT-116 cells express autocrine PG, the in situ association of PG with ANX II was demonstrated in pulldown assays. Direct binding of PG with ANX II was confirmed in an in vitro binding assay. In order to confirm a functional importance of these observations, sense and anti-sense (AS) ANX II RNA-expressing clones of intestinal epithelial (IEC-18) and human colon cancer (HCT-116) cell lines were generated. AS clones demonstrated a significant loss in the growth response to exogenous (IEC-18) and autocrine (HCT-116) PG. We have thus discovered that membrane-associated ANX II binds PG/gastrins, and partially mediates growth factor effects of the peptides.