Structure and Functional Analysis of Bone Morphogenetic Protein Receptor

我们从小鼠MC3T3-E1细胞cDNA中分离出几种新的丝氨酸/苏氨酸激酶受体cDNA，使用在tgf - β家族受体（如激活素和tgf - β II型受体）中保守序列设计的寡核苷酸引物。其中一个被命名为mtfr11的是I型受体，它可以结合BMP-2和BMP-4。我们构建了一个突变受体cDNA，预测了一个缺失整个丝氨酸/苏氨酸激酶的截断受体，并证实了显性阴性受体在培养的成骨细胞和早期胚胎中抑制BMP-2和BMP-4。此外，我们通过GS结构域Gln到Asp的点突变产生了BMP受体的组成活性形式，这被认为是BMP信号传导所必需的。我们进一步证实，突变受体传递BMP样信号独立于BMP配体。我们现在的工作重点是鉴定介导BMP信号传导的下游因子。因此，我们以BMP受体细胞质区域为诱饵，采用酵母双杂交方法筛选了人类cDNA文库。我们成功地分离了三个编码BMP受体蛋白的cdna，目前正在研究这些蛋白在信号转导中的功能。

We have isolated several novel receptor Ser/Thr kinase cDNAs from a mouse MC3T3-E1 cells cDNA using oligonucleotide primers designed in the conserved sequence among TGF-beta family receoptors such as activin and TGF-beta type II receptors. One of them designated as mTFR11turned out to be a type I class of receptor that binds both BMP-2 and BMP-4. We constructed a mutantreceptor cDNA that predicts a truncated receptor lacking entire Ser/Thr kinase and confirmed that the dominant negative receptor inhibited both BMP-2 and BMP-4 in cultured osteoblastic cells and in early embryos. Moreover, we generated constitutively active form ofthe BMP receptor by a point mutation Gln to Asp in GS domain, which is believed to be essential for BMP signaling. We further confirmed that the mutant receptor transmits BMP-like signals independent of BMP ligands.Our effort is now focused on the identification of downstream factor that mediates BMP signaling. Therefore, we have screened a human cDNA library by yeast two-hybrid method using a cytoplasmic region of the BMP receptor as a bait. Successfully, we have isolated three cDNAs that encodes proteins that binds BMP receptor and function of the proteins in the signal transduction is currently investigated.