Transforming growth factor-β induces formation of a dithiothreitol- resistant type I/type II receptor complex in live cells

Transforming growth factor-β (TGF-β) binds to and signals via two serine-threonine kinase receptors, the type I (TβRI) and type II (TβRII) receptors. We have used different and complementary techniques to study the physical nature and ligand dependence of the complex formed by TβRI and TβRII. Velocity centrifugation of endogenous receptors suggests that ligand- bound TβRI and TβRII form a heteromeric complex that is most likely a heterotetramer. Antibody-mediated immunofluorescence co-patching of epitope- tagged receptors provides the first evidence in live cells that TβRI·TβRII complex formation occurs at a low but measurable degree in the absence of ligand, increasing significantly after TGF-β binding. In addition, we demonstrate that pretreatment of cells with dithiothreitol, which inhibits the binding of TGF-~ to TβRI, does not prevent formation of the TβRI.TβRII complex, but increases its sensitivity to detergent and prevents TGF-β-activated TβRI from phosphorylating Smad3 in vitro. This indicates that either a specific conformation of the TβRI.TβRII complex, disrupted by dithiothreitol, or direct binding of TGF-β to TβRI is required for signaling.