Transforming growth factor beta (TGF-beta) is a multifunctional cytokine which controls the production of key components of the extracellular matrix and functions as a potent growth inhibitor of epithelial and endothelial cells by acting negatively on the cell cycle. TGF-beta signaling is mediated by two single pass transmembrane receptors, known as the TGF-beta type I and type II receptors, and a family of intracellular effectors, known as SMADS. Signaling in this system is initiated by assembly of a heterotetrameric signaling complex consisting of growth factor ligand and two copies each of the type I and type II receptors. At present, there is a complete lack of structural information regarding both the receptors themselves and the details of their association with this important class of growth factors. Considerable practical benefits exist in understanding the interactions which mediate growth factor-receptor assembly since TGF-beta signaling is tightly controlled, and disruption of this balance through overexpression of TGF-beta or mutations which occur naturally within the receptors or SMAD proteins has been shown to be linked to a number of pathophysiological states, including fibrotic disorders and cancer. Additionally, TGF-beta and its signaling receptors are the prototype for a large family of highly conserved growth factors and growth factor receptors, known as the TGF-beta superfamily. Many of these factors play critical roles in development and in cellular homeostasis, yet at present little information is available regarding the molecular determinants which govern ligand-receptor specificity. The primary objective of this proposal is to use a direct structure-based approach to provide definitive information regarding amino acid sequence and spatial determinants which govern ligand-receptor and receptor-receptor interactions in the heterotetrameric TGF-beta signaling complex. Experimentally, this objective will be accomplished by using solution NMR techniques to a) solve the structures of the ligand binding domains of the two receptor types and b) to delineate the determinants of specificity which govern receptor-ligand and receptor-receptor association. The model system that Dr. Hinck plans to use for these studies is the isolated ligand binding domains of the human TGF-beta type I and type II receptors. Multinuclear solution NMR techniques are ideally suited for studying this problem owing to the relatively small size of the soluble receptors, type I 11.1 kDa, type II 14.1 kDa, and recently developed E. coli expression systems. Initial experimental efforts at achieving this objective will focus on the soluble type II receptor and identification of its binding site. During the final period of the project, Dr. Hinck plans to solve the solution structure of the soluble type I receptor and to investigate its interaction with the type II receptor and TGF-beta. The general principles of receptor assembly which emerge from these studies will a) provide a framework for beginning to predict ligand-receptor specificity across the TGF-beta superfamily and b) will enable a rational approach toward intervening in the TGF-beta signaling pathway.
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