Differential Binding Activity of TGF-β Family Proteins to Select TGF-β Receptors

• Published in: The Journal of pharmacology and experimental therapeutics 2016-09, Vol.358 (3), p.423-430
• Main Authors: Khalil, Ashraf M, Dotimas, Hyna, Kahn, Julius, Lamerdin, Jane E, Hayes, David B, Gupta, Priyanka, Franti, Michael
• Format: Article
• Language: English
• Subjects: Actin-Related Protein 2 - chemistry; Actin-Related Protein 2 - metabolism; Bone Morphogenetic Proteins - metabolism; Growth Differentiation Factors - metabolism; Hep G2 Cells; Humans; Myostatin - metabolism; Protein Binding; Protein Multimerization; Protein Structure, Quaternary; Protein-Serine-Threonine Kinases - chemistry; Protein-Serine-Threonine Kinases - metabolism; Receptors, Transforming Growth Factor beta - chemistry; Receptors, Transforming Growth Factor beta - metabolism; Signal Transduction; Smad2 Protein - metabolism; Smad3 Protein - metabolism; Substrate Specificity; Transforming Growth Factor beta - metabolism
• ISSN: 1521-0103
• DOI: 10.1124/jpet.116.232322

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.