TGF-beta family members are important regulators of differentiation and tissue morphogenesis. They also act as tissue and cell specific growth inhibitors, thus making them candidate tumor suppressors. Uncovering the molecular mechanisms through which the TGF-beta family operates is fundamental to our understanding of the laws governing cell growth and differentiation. From this platform potent therapeutics for controlling or correcting disregulated cell function can be developed. Despite the wide interest in the mode of action of the TGF-beta family, little is known about the mechanisms through which they operate. Molecular cloning and characterization of both the type I and type II receptors of TGF-beta family members revealed that heteromeric complex of type I and type II receptors are required for signaling. Recent evidence suggests that the TGF-beta type II receptor phosphorylates the type I receptor, which may then be responsible for activating downstream signaling. By using the yeast two-hybrid system, the immunophilin FKBP12 was isolated as a specific interactor for the cytoplasmic domains of virtually all type I receptors. The specificity of the interaction has now been confirmed in yeast, in vitro and most recently in mammalian cell lines such as COS and mink Mv1Lu cells. This proposal aims to characterize the molecular details of the interaction between FKBP12 and the TGF-beta type I receptor to identify the functional significance of the interaction, and to understand the role of FKBP12 in TGF-beta type I receptor-mediated signaling. Specifically, the following studies are proposed. 1) Detailed biochemical analysis of the interaction will be carried out in mammalian cells to study how ligand binding, type I receptor phosphorylation or type I receptor kinase activity affects the interaction between FKBP12 and the type I receptor. The possible activity of FKBP12 in regulating type I receptor kinase activity will also be analyzed. 2) To address the functional significance of the interaction, a combination of yeast genetics, molecular biological biochemical approaches will be used to disrupt the interaction in TGF-beta responsive cell lines. The role of the peptidyl-prolyl-isomerase activity of FKBP12 in TGF-beta signaling will also be studied. 3) Structural analysis of FKBP12/type I receptor interaction will be carried out (collaboratively) in order to unequivocally identify the interaction domains on both proteins. These studies will provide a foundation for subsequent specific molecular engineering and structure-based drug designs. 4) Downstream interactors of FKBP12 or FKBP12/type I receptor complex will be identified in order to further our understanding of the function of FKBP12 in TGF-beta type l receptor-mediated signaling. The studies proposed here will serve as a paradigm for future studies on other interactors of the TGF-beta type I receptor family.
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