Mazerbourg Sabine, Sangkuhl Katrin, Luo Ching-Wei, Sudo Satoko, Klein Cynthia, Hsueh Aaron J W
Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, California 94305-5317, USA.
J Biol Chem. 2005 Sep 16;280(37):32122-32. doi: 10.1074/jbc.M504629200. Epub 2005 Jul 27.
There are more than 30 human transforming growth factor beta/bone morphogenetic protein/growth differentiation factor (TGFbeta/BMP/GDF)-related ligands known to be important during embryonic development, organogenesis, bone formation, reproduction, and other physiological processes. Although select TGFbeta/BMP/GDF proteins were found to interact with type II and type I serine/threonine receptors to activate downstream Smad and other proteins, the receptors and signaling pathways for one-third of these TGFbeta/BMP/GDF paralogs are still unclear. Based on a genomic analysis of the entire repertoire of TGFbeta/BMP/GDF ligands and serine/threonine kinase receptors, we tested the ability of three orphan BMP/GDF ligands to activate a limited number of phylogenetically related receptors. We characterized the dimeric nature of recombinant GDF6 (also known as BMP13), GDF7 (also known as BMP12), and BMP10. We demonstrated their bioactivities based on the activation of Smad1/5/8-, but not Smad2/3-, responsive promoter constructs in the MC3T3 cell line. Furthermore, we showed their ability to induce the phosphorylation of Smad1, but not Smad2, in these cells. In COS7 cells transfected with the seven known type I receptors, overexpression of ALK3 or ALK6 conferred ligand signaling by GDF6, GDF7, and BMP10. In contrast, transfection of MC3T3 cells with ALK3 small hairpin RNA suppressed Smad signaling induced by all three ligands. Based on the coevolution of ligands and receptors, we also tested the role of BMPRII and ActRIIA as the type II receptor candidates for the three orphan ligands. We found that transfection of small hairpin RNA for BMPRII and ActRIIA in MC3T3 cells suppressed the signaling of GDF6, GDF7, and BMP10. Thus, the present approach provides a genomic paradigm for matching paralogous polypeptide ligands with a limited number of evolutionarily related receptors capable of activating specific downstream Smad proteins.
已知有30多种人类转化生长因子β/骨形态发生蛋白/生长分化因子（TGFβ/BMP/GDF）相关配体在胚胎发育、器官形成、骨形成、生殖及其他生理过程中发挥重要作用。尽管已发现某些TGFβ/BMP/GDF蛋白可与II型和I型丝氨酸/苏氨酸受体相互作用以激活下游的Smad及其他蛋白，但这些TGFβ/BMP/GDF旁系同源物中三分之一的受体和信号通路仍不清楚。基于对TGFβ/BMP/GDF配体和丝氨酸/苏氨酸激酶受体全部组成的基因组分析，我们测试了三种孤儿BMP/GDF配体激活有限数量的系统发育相关受体的能力。我们对重组GDF6（也称为BMP13）、GDF7（也称为BMP12）和BMP10的二聚体性质进行了表征。我们基于在MC3T3细胞系中激活Smad1/5/8响应性启动子构建体（而非Smad2/3响应性启动子构建体）证明了它们的生物活性。此外，我们展示了它们在这些细胞中诱导Smad1而非Smad2磷酸化的能力。在转染了七种已知I型受体的COS7细胞中，ALK3或ALK6的过表达赋予了GDF6、GDF7和BMP10配体信号传导能力。相反，用ALK3小发夹RNA转染MC3T3细胞可抑制所有三种配体诱导的Smad信号传导。基于配体和受体的共同进化，我们还测试了BMPRII和ActRIIA作为这三种孤儿配体的II型受体候选者的作用。我们发现，在MC3T3细胞中转染针对BMPRII和ActRIIA的小发夹RNA可抑制GDF6、GDF7和BMP10的信号传导。因此，本方法为将旁系同源多肽配体与能够激活特定下游Smad蛋白的有限数量的进化相关受体进行匹配提供了一种基因组范式。
