Mutational Analysis of a Transforming Growth Factor-β Receptor Binding Site

Transforming growth factor-beta s (TGF-beta 1, -beta 2, -beta 3) are important regulators of cell growth and differentiation which share approximately 70% identical amino acids. Using LS513 colorectal cells, which are growth inhibited by TGF-beta 1 (ED50 of 100 pM), but are refractory to TGF-beta 2 (ED50 of 50,000 to 100,000 pM), we have determined that amino acids 92-98 of TGF-beta specify growth inhibition. The chimeric protein TGF-beta 1/beta 2(92-98), in which amino acids 92-98 of TGF-beta 1 were exchanged for the corresponding amino acids of TGF-beta 2, was indistinguishable from TGF-beta 2 at inhibiting growth of LS513 cells. In contrast, both TGF-beta 1/beta 2(92-95) and TGF-beta 1/beta 2(94-98) inhibited the growth of LS513 cells with an ED50 of approximately 1000 pM. TGF-beta 1/beta 2(95-98), in which amino acids 95-98 of TGF-beta 1 have been replaced with the corresponding amino acids of TGF-beta 2, had full activity and was indistinguishable from TGF-beta 1. Receptor cross-linking experiments demonstrated that binding of the chimeras to the type I and type II receptors of LS513 cells was consistent with their biological activity. TGF-beta 1/beta 2(95-98), TGF-beta 1/beta 2(92-95) and TGF-beta 1/beta 2(94-98) were each similar to TGF-beta 2 in that they failed to bind to the soluble Type II receptor in a solid-phase assay. These results demonstrate that amino acids 92-98 are involved in the interaction between TGF-beta and its signaling receptors and they show that modest changes within this region can substantially alter biological response.