Distinct oligomeric states of SMAD proteins in the transforming growth factor-beta pathway

Protein interactions are critical for the function of SMADs as mediators of transforming growth factor-beta (TGF-beta) signals. TGF-beta receptor phos phorylation of SMAD2 or SMAD3 causes their association with SMAD4 and accum ulation in the nucleus where the SMAD complex binds cofactors that determin e the choice of target genes. We provide evidence that in the basal state, SMADs 2, 3, and 4 form separate, strikingly different complexes. SMAD2 is f ound mostly as monomer, whereas the closely related SMAD3 exists in multipl e oligomeric states. This difference is due to a unique structural element in the MH1 domain of SMAD2 that inhibits protein-protein interactions in th e basal state. In contrast to SMAD2 and SMAD3, SMAD4 in the basal state is found mostly as a homo-oligomer, most likely a trimer. Upon cell stimulatio n with TGF-beta, SMAD proteins become engaged in a multitude of complexes r anging in size from SMAD2-SMAD4 heterodimers to assemblies of >650 kDa, The latter display the highest DNA binding affinity for the TGF-beta -response elements of JUNB and collagen 7. These observations, all validated with en dogenous SMAD proteins, modify previous models regarding the assembly and a ctivity of SMAD complexes in the TGF-beta pathway.