Binding affinity of transforming growth factor-β for its type II receptor is determined by the C-terminal region of the molecule

Abstract

Transforming growth factor-β (TGF-β) isoforms have differential binding affinities for the TGF-β type II receptor (TβRII). In most cells, TGF-β1 and TGF-β3 bind to TβRII with much higher affinity than TGF-β2. Here, we report an analysis of the effect of TGF-β structure on its binding to TβRII by using TGF-β mutants with domain deletions, amino acid replacements, and isoform chimeras. Examination of the binding of TGF-β mutants to the recombinant extracellular domain of TβRII by a solid-phase TGF-β/TβRII assay demonstrated that only those TGF-β mutants containing the C terminus of TGF-β1 (TGF-β1-(Δ69-73), TGF-β1-(Trp71), and TGF- β2/β1-(83112)) bind with high affinity to TβRII, similar to native TGF- β1. Moreover, replacement of only 6 amino acids in the C terminus of TGF- β1 with the corresponding sequence of TGF-β2 (TGF-β1/β2-(91-96)) completely eliminated the high affinity binding of TGF-β1. Proliferation of fetal bovine heart endothelial (FBHE) cells was inhibited to a similar degree by all of the TGF-β mutants. However, recombinant soluble TβRII blocked the inhibition of FBHE cell proliferation induced by TGF-β mutants retaining the C terminus of TGF-β1, consistent with the high binding affinity between these TGF-β molecules and TβRII. It was further confirmed that the TGF-β2 mutant with its C terminus replaced by that of TGF-β1 (TGF-β2/β1-(83- 112)) competed as effectively as TGF-β1 with 125I-TGF-β1 for binding to membrane TβRI and TβRH on FBHE cells. These observations clearly indicate that the domain in TGF-β1 responsible for its high affinity binding to TβRII, both the soluble and membrane-bound forms, is located at C terminus of the molecule.