Assembly of TβRI:TβRII:TGFβ Ternary Complex in vitro with Receptor Extracellular Domains is Cooperative and Isoform-dependent

Transforming growth factor-β (TGFβ) isoforms initiate signaling by assembling a heterotetrameric complex of paired type I (TβRI) and type II (TβRII) receptors on the cell surface. Because two of the ligand isoforms (TGFβs 1, 3) must first bind TβRII to recruit TβRI into the complex, and a third (TGFβ2) requires a co-receptor, assembly is known to be sequential, cooperative and isoform-dependent. However the source of the cooperativity leading to recruitment of TβRI and the universality of the assembly mechanism with respect to isoforms remain unclear. Here, we show that the extracellular domain of TβRI (TβRI-ED) binds in vitro with high affinity to complexes of the extracellular domain of TβRII (TβRII-ED) and TGFβs 1 or 3, but not to either ligand or receptor alone. Thus, recruitment of TβRI requires combined interactions with TβRII-ED and ligand, but not membrane attachment of the receptors. Cell-based assays show that TβRI-ED, like TβRII-ED, acts as an antagonist of TGFβ signaling, indicating that receptor–receptor interaction is sufficient to compete against endogenous, membrane-localized receptors. On the other hand, neither TβRII-ED, nor TβRII-ED and TβRI-ED combined, form a complex with TGFβ2, showing that receptor–receptor interaction is insufficient to compensate for weak ligand–receptor interaction. However, TβRII-ED does bind with high affinity to TGFβ2-TM, a TGFβ2 variant substituted at three positions to mimic TGFβs 1 and 3 at the TβRII binding interface. This proves both necessary and sufficient for recruitment of TβRI-ED, suggesting that the three different TGFβ isoforms induce assembly of the heterotetrameric receptor complex in the same general manner.