TGF?s and other structurally related proteins of the TGF? superfamily, such as BMPs, GDFs, and activins, are secreted signaling proteins that regulate many essential processes, ranging from establishment of the body plan during embryogenesis to regulation of the adaptive immune system. The importance of the more than 30 superfamily members is demonstrated by the many human diseases that result from dysregulation of the pathway, including skeletal and cardiac disorders, infertility, and cancer. The development of effective therapies for treating pathway-associated diseases requires knowledge of the interactions that govern ligand activities - this includes interactions with the signaling receptors, but as well with the many accessory proteins that bind ligands and regulate ligand accessibility. The objective of this project is to capitalize upon our current understanding of how TGF?s bind their signaling receptors to investigate how betaglycan, a membrane- anchored accessory binding proteolgycan, functions on the one hand to promote TGF? signaling, and on the other to inhibit activin signaling. The potentiation of TGF? signaling by betaglycan is thought to arise from its ability to bind and localize TGF? ligands on the cell surface. To investigate this, we will determine the structures of betaglycan's two independent TGF? binding domains, BGE and BGU, bound to TGF? using crystallography, which is ideal for these purposes, as BGE and BGU bind and form highly stable complexes with TGF?. The mechanisms for potentiation of receptor binding suggested by the structure of the complexes will be tested in functional assays. The inhibition of activin signaling by betaglycan is thought to be mediated by sequestration of the activin type II receptor, ActRII or the close homolog ActRIIb, in a ternary complex with betaglycan and inhibin A, a heterodimeric ligand with an activin ? subunit that binds the activin type II receptor, ActRII/ActRIIb, and an activin ? subunit that binds BGU, betaglycan's C-terminal binding domain. To investigate this, we will first determine how activins bind their type I receptor, ActRIb. These studies will be carried out using NMR as ActRIb binds activin A weakly. This will be further investigated by determining the structure of inhibin A bound to BGU using crystallography, which is ideal for these purposes as BGU binds and forms a highly stable complex with inhibin A. The mechanisms for antagonism of activin by inhibin A suggested by the structure of the complexes will be tested in functional assays. The feasibility of the proposed studies is demonstrated by diffracting crystals obtained for the BGE:T?RII:TGF? complex, the ability to isolate the BGUC:TGF? complex, and NMR signal assignments for ActRIb. The determination of these structures will provide an important missing link in our understanding of how one of the accessory proteins of the superfamily, betaglycan, influences downstream signaling induced by TGF?s and activin. The structures will also aid in the development of novel targeted-therapies for treatment of diseases and disorders, such as cancer and infertility, caused by aberrant TGF? and activin signaling.
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