Transforming Growth Factor-β (TGF-β) family ligands are key regulators of multiple cellular processes including cell proliferation, differentiation, and death. Dysregulation of TGF-β family signaling thus contributes to many human diseases, including cancers, fibrosis, and musculoskeletal disorders. Because of its roles in human diseases, understanding the mechanism and regulation of TGF-β family signal transduction is essential and will help the development of new therapeutic agents that could be used to target the family for treating a number of devastating diseases. The basic mechanisms of TGF-β family action are well established. A dimeric ligand binds two 'type I' and two 'type II' receptors to form a hexameric complex. Assembly of the ligand-receptor complex in the extracellular space leads to phosphorylation of SMAD transcription factors in the intracellular space, their translocation to the nucleus, and expression of target genes. However, beyond ligands and receptors, many additional factors contribute critically to physiological TGF-β family signaling, including membrane-bound co-receptors, secreted inhibitors, and other ligands that are present on or near the surface of a cell. Elucidating the molecular interplay of all the components that form the TGF-β signal transduction system of a particular cell type or tissue is essential for understanding TGF-β signaling in a cellular context. The goal of my thesis was to investigate the extracellular regulation mechanisms of the TGF-β family ligands, Nodal and Activin using biophysical approaches and to define the physiological consequences using cell-based approaches. In the first chapter of my dissertation, I introduced the TGF-β family and described how I expressed and purified TGF-β family ligands, receptors, and extracellular regulators. I also introduced a powerful technique called Surface Plasmon Resonance (SPR) that I used to determine the binding modes between TGF-β family members. In the second chapter of my dissertation, I elucidated the molecular function of TGF-β family signaling co-receptor Cripto-1 and its homolog. I demonstrated that soluble Cripto-1 inhibits ligand induced signaling, but the membrane-anchored form potentiates the signaling, suggesting an extracellular capture and endocytic trafficking mechanism. In the third chapter of my dissertation, I characterized the function of the TGF-β family ligand Nodal and its natural inhibitor Cerberus in breast cancer cell lines. I found that Cerberus significantly suppresses migration, invasion, and colony formation of Nodal expressing breast cancer cells. In the fourth chapter of my dissertation, I defined the function of human Cerberus in TGF-β family signaling. I showed that full-length and short forms of human Cerberus share TGF-β family ligand binding and inhibition mechanism, but only full-length Cerberus suppresses MDA-MB-231 breast cancer cell migration. In the fifth chapter of my dissertation, I evaluated how the population of different ligands affects signaling outcomes. I demonstrated that high affinity TGF-β family ligands compete with low affinity ligands for receptor binding and antagonize low affinity ligand signaling. In the sixth chapter of my dissertation, I discussed the possible future directions for this project. Collectively, the results of these experiments provide new information about TGF-β family signaling and regulation, and potentially lead to evaluation of new therapeutics targeting family members in different diseases.
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