Lu Ganwei, Maeda Hidefumi, Reddy Sakamuri V, Kurihara Noriyoshi, Leach Robin, Anderson Judith L, Roodman G David
Medicine-Hematology/Oncology, University of Pittsburgh, Pittsburgh, Pennsylvania 15240, USA.
J Biol Chem. 2006 Oct 13;281(41):30542-50. doi: 10.1074/jbc.M607072200. Epub 2006 Aug 8.
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. 125I-Labeled annexin II binding assays with PSV10 cells demonstrated that there was a single class of annexin II receptors with a Kd of 5.79 nm and Bmax of 2.13 x 10(5) receptors/cell. Annexin III or annexin V did not bind this receptor. Using 125I-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.
膜联蛋白II是一种异源四聚体，由两个11 kDa（p11）亚基和两个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，其受体尚不清楚。我们发现膜联蛋白II与正常原代人骨髓基质细胞以及佩吉特骨髓来源的PSV10基质细胞系结合，以诱导破骨细胞形成。用PSV10细胞进行的125I标记膜联蛋白II结合试验表明，存在一类膜联蛋白II受体，其解离常数（Kd）为5.79 nM，最大结合容量（Bmax）为2.13×10⁵个受体/细胞。膜联蛋白III或膜联蛋白V不与该受体结合。利用125I标记的膜联蛋白II结合筛选用人骨髓cDNA表达文库转染的NIH3T3细胞，我们鉴定出一个假定的膜联蛋白II受体克隆，当在HEK 293细胞中表达时，它编码一种新的26 kDa I型膜受体蛋白。用克隆的膜联蛋白II受体cDNA转化的HEK 293细胞对膜联蛋白II的结合亲和力与在PSV10细胞中观察到的相似。用生物素化的膜联蛋白II和完整的PSV10细胞进行化学交联实验，在蛋白质印迹分析中鉴定出一条55 kDa的条带，它与抗p11抗体和链霉亲和素反应，但不与抗p36抗体反应。针对假定的重组膜联蛋白II受体产生的兔多克隆抗体也识别在PSV10细胞中检测到的相同的26 kDa蛋白条带。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，表明该受体介导膜联蛋白II对破骨细胞形成的作用。
