The L3 loop: a structural motif determining specific interactions between SMAD proteins and TGF‐β receptors

Signal transduction specificity in the transforming growth factor‐β (TGF‐β) system is determined by ligand activation of a receptor complex which then recruits and phosphorylates a subset of SMAD proteins including Smads 1 and 2. These then associate with Smad4 and move into the nucleus where they regulate transcription. We have identified a discrete surface structure in Smads 1 and 2 that mediates and specifies their receptor interactions. This structure is the L3 loop, a 17 amino acid region that protrudes from the core of the conserved SMAD C‐terminal domain. The L3 loop sequence is invariant among TGF‐β‐ and bone morphogenetic protein (BMP)‐activated SMADS, but differs at two positions between these two groups. Swapping these two amino acids in Smads 1 and 2 induces a gain or loss, respectively, in their ability to associate with the TGF‐β receptor complex and causes a switch in the phosphorylation of Smads 1 and 2 by the BMP and TGF‐β receptors, respectively. A full switch in phosphorylation and activation of Smads 1 and 2 is obtained by swapping both these two amino acids and four amino acids near the C‐terminal receptor phosphorylation sites. These studies identify the L3 loop as a determinant of specific SMAD–receptor interactions, and indicate that the L3 loop, together with the C‐terminal tail, specifies SMAD activation.