Khalil Ashraf M, Dotimas Hyna, Kahn Julius, Lamerdin Jane E, Hayes David B, Gupta Priyanka, Franti Michael
Boehringer Ingelheim Pharmaceuticals, Inc. (A.M.K., J.K., D.B.H., P.G., M.F.), Ridgefield, Connecticut; and DiscoverX Corporation (H.D., J.E.L.), Fremont, California.
Boehringer Ingelheim Pharmaceuticals, Inc. (A.M.K., J.K., D.B.H., P.G., M.F.), Ridgefield, Connecticut; and DiscoverX Corporation (H.D., J.E.L.), Fremont, California
J Pharmacol Exp Ther. 2016 Sep;358(3):423-30. doi: 10.1124/jpet.116.232322. Epub 2016 Jun 23.
Growth differentiation factor-11 (GDF11) and myostatin (MSTN) are highly related transforming growth factor-β (TGF-β) ligands with 89% amino acid sequence homology. They have different biologic activities and diverse tissue distribution patterns. However, the activities of these ligands are indistinguishable in in vitro assays. SMAD2/3 signaling has been identified as the canonical pathway for GDF11 and MSTN, However, it remains unclear which receptor heterodimer and which antagonists preferentially mediate and regulate signaling. In this study, we investigated the initiation and regulation of GDF11 and MSTN signaling at the receptor level using a novel receptor dimerization detection technology. We used the dimerization platform to link early receptor binding events to intracellular downstream signaling. This approach was instrumental in revealing differential receptor binding activity within the TGF-β family. We verified the ActR2b/ALK5 heterodimer as the predominant receptor for GDF11- and MSTN-induced SMAD2/3 signaling. We also showed ALK7 specifically mediates activin-B signaling. We verified follistatin as a potent antagonist to neutralize both SMAD2/3 signaling and receptor dimerization. More remarkably, we showed that the two related antagonists, growth and differentiation factor-associated serum protein (GASP)-1 and GASP2, differentially regulate GDF11 (and MSTN) signaling. GASP1 blocks both receptor dimerization and downstream signaling. However, GASP2 blocks only downstream signaling without interference from receptor dimerization. Our data strongly suggest that physical binding of GDF11 (and MSTN) to both ActR2b and ALK5 receptors is required for initiation of signaling.
生长分化因子11（GDF11）和肌肉生长抑制素（MSTN）是高度相关的转化生长因子-β（TGF-β）配体，氨基酸序列同源性达89%。它们具有不同的生物学活性和多样的组织分布模式。然而，在体外试验中这些配体的活性难以区分。SMAD2/3信号通路已被确定为GDF11和MSTN的经典信号通路，然而，尚不清楚哪种受体异二聚体以及哪种拮抗剂优先介导和调节信号传导。在本研究中，我们使用一种新型受体二聚化检测技术在受体水平研究了GDF11和MSTN信号传导的起始和调节。我们利用二聚化平台将早期受体结合事件与细胞内下游信号传导联系起来。这种方法有助于揭示TGF-β家族内不同的受体结合活性。我们证实了ActR2b/ALK5异二聚体是GDF11和MSTN诱导的SMAD2/3信号传导的主要受体。我们还表明ALK7特异性介导激活素B信号传导。我们证实卵泡抑素是一种有效的拮抗剂，可中和SMAD2/3信号传导和受体二聚化。更值得注意的是，我们表明两种相关的拮抗剂，生长和分化因子相关血清蛋白（GASP）-1和GASP2，对GDF11（和MSTN）信号传导有不同的调节作用。GASP1阻断受体二聚化和下游信号传导。然而，GASP2仅阻断下游信号传导，而不干扰受体二聚化。我们的数据强烈表明，GDF11（和MSTN）与ActR2b和ALK5受体的物理结合是信号传导起始所必需的。
