Cloning and characterization of the annexin II receptor on human marrow stromal cells
Cloning and characterization of the annexin II receptor on human marrow stromal cells
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DOI：
10.1074/jbc.m607072200
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发表时间：
2006-10-13
影响因子：
4.8
通讯作者：
Roodman, G. David
中科院分区：
文献类型：
--
作者：
Lu, Ganwei;Maeda, Hidefumi;Roodman, G. David
膜联蛋白II是一种异源四聚体，由两个11-kDa（p11）和两个36-kDa（p36）亚基组成，由破骨细胞产生并刺激破骨细胞形成。然而，它的受体是未知的。我们发现，膜联蛋白II结合正常的原代人骨髓基质细胞和佩吉特骨髓来源的PSV 10基质细胞系，诱导破骨细胞的形成。用PSV 10细胞进行的I-125标记的膜联蛋白II结合试验表明，存在单一类别的膜联蛋白II受体，Kd为5.79 nM，B-max为2.13 x 10（5）受体/细胞。膜联蛋白III或膜联蛋白V不结合该受体。使用I-125标记的膜联蛋白II结合筛选转染人骨髓cDNA表达文库的NIH3T3，我们确定了一个推定的膜联蛋白II受体克隆，其编码一种新的26 kDa的I型膜受体蛋白在HEK 293细胞中表达时。用克隆的膜联蛋白II受体cDNA转化的HEK 293细胞显示出与在PSV 10细胞中观察到的膜联蛋白II相似的结合亲和力。用生物素化膜联蛋白II和完整的PSV 10细胞进行的化学交联实验在Western印迹分析中鉴定出一个55 kDa的条带，该条带与抗p11抗体和链霉亲和素反应，但不与抗p36抗体反应。兔多克隆抗体提出了对推定的重组膜联蛋白II受体也承认相同的26 kDa的蛋白质带中检测到的PSV 10细胞。重要的是，膜联蛋白II受体抗体剂量依赖性地阻断膜联蛋白II对人破骨细胞形成的刺激作用，表明受体介导膜联蛋白II对破骨细胞形成的作用。
Annexin II is a heterotetramer, consisting of two 11-kDa (p11) and two 36-kDa (p36) subunits, that is produced by osteoclasts and stimulates osteoclast formation. However, its receptor is unknown. We showed that annexin II binds to normal primary human marrow stromal cells and the Paget's marrow-derived PSV10 stromal cell line to induce osteoclast formation. I-125-Labeled annexin II binding assays with PSV10 cells demonstrated that there was a single class of annexin II receptors with a K-d of 5.79 nM and B-max of 2.13 x 10(5) receptors/cell. Annexin III or annexin V did not bind this receptor. Using I-125-labeled annexin II binding to screen NIH3T3 transfected with a human marrow cDNA expression library, we identified a putative annexin II receptor clone, which encoded a novel 26-kDa type I membrane receptor protein when expressed in HEK 293 cells. HEK 293 cells transformed with the cloned annexin II receptor cDNA showed a similar binding affinity to annexin II as that observed in PSV10 cells. Chemical cross-linking experiments with biotinylated annexin II and intact PSV10 cells identified a 55-kDa band on Western blot analysis that reacted with both an anti-p11 antibody and streptavidin but not anti-p36 antibody. A rabbit polyclonal antibody raised against the putative recombinant annexin II receptor also recognized the same 26-kDa protein band detected in PSV10 cells. Importantly, the annexin II receptor antibody dose-dependently blocked the stimulatory effects of annexin II on human osteoclast formation, demonstrating that the receptor mediates the effects of annexin II on osteoclast formation.