Investigation of TGF-B Ligand Signaling Through Activation of Specific Receptors

Abstract

TGF-b proteins are a superfamily of secreted, extracellular signaling ligands which bind and activate two type I and two type II receptors to modulate transcriptional output in nearly every biological process. It is not yet understood how discrete ligand signaling occurs despite overlapping receptor utilization by the 33 unique TGF-b ligands. Growth differentiation factors-8 (GDF8) and -11 (GDF11) are two TGF-b ligands that share 90% sequence identity in their mature signaling domains and are known to utilize similar receptors yet are thought to play distinct biological roles. Our study aimed to determine if the potency differences between GDF8 and GDF11 are mediated through specific receptor pairings leading to activation of intracellular SMAD molecules. We used CRISPR technology to create type I receptor knock out HEK293 cell lines, ultimately creating two cell lines which lacked ALK4 and ALK5. We used these cell lines to determine the half maximal effective concentration (EC50) of GDF8 and GDF11 by assessing activation of a SMAD3-responsive luciferase reporter. We assessed activation of the knockout cell lines with GDF8 and GDF11 and found that ALK4 and ALK5 may independently mediate the potency differences previously discovered. Because aberrant TGF-b signaling often results in a wide variety of pathologies, future work is important to continue to unravel the mechanisms governing TGF-b signaling.