The type V TGF-beta receptor (TbetaR) is a 400-kDa acidotropic Ser/Thr- specific protein kinase which co-expresses with the type I, type II and type III TGF-beta receptors (TbetaR-I, TbetaR-II and TbetaR-III) in most cell types. Its selective absence in most epithelial tumor cells studied suggests that loss of TbetaR-V expression may be a step toward tumorigenesis of epithelial cells. The novel finding that TbetaR-V is the receptor of insulin-like growth factor binding protein 3 (IGFBP-3) which mediates the IGF-independent growth inhibition has provided the strongest evidence to support the function of TbetaR-V in mediating growth inhibition. We have recently found that the IGFBP-3 growth inhibition is reversed by insulin/IGF-1 and TGF-beta growth inhibition is partially reversed by insulin/IGF-I in the presence of a cyclic RGD peptide. These observations have uncovered intriguing """"""""cross-talk"""""""" interactions of TbetaR-V, TbetaR-I/TbetaR-II and insulin receptor/IGF-I receptor signaling pathways. The broad goal of the proposed research is to define the mechanism of TbetaR-V-mediated growth inhibition and to test the hypothesis that TbetaR-V is the product of a tumor suppressor gene. The proposal has three specific aims: 1) To determine the mechanisms by which insulin/IGF-I block the IGFBP-3 growth inhibition mediated by TbetaR-V. We will determine the roles of IRS-1 and IRS-2 in this insulin/IGF-I effect in DR26 cells by over-expressing IRS-1/IRS-2, by blocking IRS-1/IRS-2 expression with antisense mRNA and by cell biological methods. 2) To characterize the TbetaR-V-mediated signaling using IGFP-3 as a specific ligand. We will determine the effects of IGFBP-3 on TbetaR-V/TbetaR-I heterocomplex formation, phosphorylation/complex formation/nuclear translocation of Smad2/Smad3/Smad4, and the expressions and activities of the key components regulating cell cycle transmission from G/1-to-S phase using cell biological methods. 3) To clone, sequence and express TbetaR-V cDNA. We will clone the kinase domain and full-length cDNA of TbetaR-V from liver cDNA libraries by PCR and immunoscreening and express the chimera of TbetaR-I and TbetaR-II extracellular domains/TbetaR-V kinase domain and full-length TbetaR-V cDNA in carcinoma cells lacking TbetaR-V in an attempt to restore TGF-beta growth inhibition and to reverse the transformed phenotype of these carcinoma cells.
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