Formation of a stable heterodimer between Smad2 and Smad4

Smad proteins mediate transforming growth factor beta signaling from the ce ll membrane to the nucleus. Upon phosphorylation by the activated receptor kinases, the receptor-regulated Smad, such as Smad2, forms a heterocomplex with the co-mediator Smad, Smad4. This heterocomplex is then translocated i nto the nucleus, where it associates with other transcription factors and r egulates expression of ligand-responsive genes. The stoichiometry between r eceptor-regulated Smad and co-mediator Smad is important for understanding the molecular mechanisms of the signaling process. Using purified recombina nt proteins, we demonstrate that Smad2 and Smad4 form a stable heterodimer and that the Smad4 activation domain is important for the formation of this complex. Many tumor-derived missense mutations disrupt the formation of th is heterocomplex in in vitro interaction assays. Mapping these mutations on to the structures of Smad4 and Smad2 identifies a symmetric interface betwe en these two Smad proteins. importantly, two previous models on the formati on of a heterocomplex are incompatible with our observations and other repo rted evidence.