Assembly of TβRI:TβRII:TGFβ ternary complex in vitro with receptor extracellular domains is cooperative and isoform-dependent

Transforming growth factor-beta (TGF beta) isoforms initiate signaling by assembling a heterotetrameric complex of paired type I (T beta RI) and type II (T beta RII) receptors on the cell surface. Because two of the ligand isoforms (TGF beta s 1, 3) must first bind T beta RII to recruit T beta RI into the complex, and a third (TGF beta 2) requires a co-receptor, assembly is known to be sequential, cooperative and isoform-dependent. However the source of the cooperativity leading to recruitment of T beta RI and the universality of the assembly mechanism with respect to isoforms remain unclear. Here, we show that the extracellular domain of T beta RI (T beta RI-ED) binds in vitro with high affinity to complexes of the extracellular domain of T beta RII (T beta RII-ED) and TGF beta s I or 3, but not to either ligand or receptor alone. Thus, recruitment of T beta RI requires combined interactions with T beta RII-ED and ligand, but not membrane attachment of the receptors. Cell-based assays show that T beta RI-ED, like T beta RII-ED, acts as an antagonist of TGF beta signaling, indicating that receptor-receptor interaction is sufficient to compete against endogenous, membrane-localized receptors. On the other hand, neither T beta RII-ED, nor T beta RII-ED and T beta RI-ED combined, form a complex with TGF beta 2, showing that receptor-receptor interaction is insufficient to compensate for weak ligand-receptor interaction. However, T beta RII-ED does bind with high affinity to TGF beta 2-TM, a TGF beta 2 variant substituted at three positions to mimic TGF beta s 1 and 3 at the T beta RII binding interface. This proves both necessary and sufficient for recruitment of T beta RI-ED, suggesting that the three different TGF beta isoforms induce assembly of the heterotetrameric receptor complex in the same general manner. (c) 2005 Elsevier Ltd. All rights reserved.