1521-0103/358/3/423–430$25.00 http://dx.doi.org/10.1124/jpet.116.232322 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 358:423–430, September 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Differential Binding Activity of TGF-b Family Proteins to Select TGF-b Receptors Ashraf M. Khalil, Hyna Dotimas, Julius Kahn, Jane E. Lamerdin, David B. Hayes, Priyanka Gupta, and Michael Franti 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 Received January 20, 2016; accepted June 22, 2016 ABSTRACT Growth differentiation factor-11 (GDF11) and myostatin (MSTN) are highly related transforming growth factor-b (TGF-b) ligands with 89% amino acid sequence homology. They have different biologic activities and diverse tissue distribution patterns. How- ever, 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-b family. We verified the ActR2- b/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, differ- entially 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. Introduction The transforming growth factor-b (TGF-b) superfamily proteins are potent regulatory cytokines. In addition to their vital role in early development and homeostasis (Wu and Hill, 2009), they also play important roles in self-tolerance and autoimmunity (Li et al., 2006). TGF-b proteins selectively signal through multiple and variable cell surface serine/th- reonine kinase receptors (de Caestecker, 2004). The Mad gene from Drosophila 1 and the related Sma genes from Caeno- rhabditis elegans 2 (Smad) have been genetically implicated in signaling by members of the bone morphogenetic protein (BMP) group (Liu et al., 1996). The biologically active dimeric mature form of growth differentiation factor-11 (GDF11), myostatin (MSTN), and activin (Huang et al., 2011) can induce signal by initially binding to predominantly ActR2b, a type II receptor. This, in turn, forms a heterodimeric complex with one or more type I receptors, mainly, ALK4, ALK5, and ALK7. Phosphorylation of type I receptors by the ActR2b receptor will recruit and phosphorylate receptor-regulated Smad2 and receptor-regulated Smad3. The activated receptor-regulated Smad2/3 forms a complex with Smad4, which translocates to the nucleus to regulate expression of downstream genes (Dennler et al., 1998). The nature of the ligand: the receptor binding that drives ActR2b to preferentially pair with multi- ple type I receptors has not been fully elucidated. In this study, we focused on the two closely related TGF-b family members, GDF11 and MSTN, which have been reported in multiple age-related disorders (Loffredo et al., 2013; Katsimpardi et al., 2014; Sinha et al., 2014; Egerman et al., 2015). They share the same signaling pathway and 89% se- quence structure homology (McPherron et al., 1997; Nakashima et al., 1999). The similarity between the two ligands led to an indistinguishable in vitro functional activity (Egerman et al., 2015). However, studies of mRNA expression have revealed distinct in vivo tissue distribution patterns indicating diverse biologic functions. Expression of MSTN is mostly limited to skeletal muscles and a high level is correlated with inhibition of muscle growth (McPherron et al., 1997; Zimmers et al., 2002). GDF11 is widely expressed and involved in the development of multiple tissues (Wu et al., 2003; Harmon et al., 2004; Kim et al., 2005). Levels of circulating GDF11 decline in aged mice leading to multiple age-related disorders (Loffredo et al., 2013; Katsimpardi et al., 2014; Sinha et al., 2014; Egerman et al., 2015). However, a conflicting report showed that GDF11 levels increase with age in rats and humans, and correlate with dx.doi.org/10.1124/jpet.116.232322. ABBREVIATIONS: BMP, bone morphogenetic protein; EA, enzyme acceptor; FBS, fetal bovine serum; GASP, growth and differentiation factor–associated serum protein; GDF11, growth differentiation factor-11; HepG2, human liver cancer cell line; MSTN, myostatin; PK, Prolink; TGF-b, transforming growth factor-b; U-2 OS, human osteosarcoma cell line. 423 at ASPET Journals on June 13, 2020 jpet.aspetjournals.org Downloaded from