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

Abstract

Transforming growth factor-βs (TGF-β1,-β2,-β3) 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-β 1 (ED₅₀ of 100 pM), but are refractory to TGF-β2 (ED₅₀ of 50,000 to 100,000 pM), we have determined that amino acids 92-98 of TGF-β specify growth inhibition. The chimeric protein TGF-β1/β2(92-98), in which amino acids 92-98 of TGF-β 1 were exchanged for the corresponding amino acids of TGF-β2, was indistinguishable from TGF-β2 at inhibiting growth of LS513 cells. In contrast, both TGF-β1/β2(92-95) and TGF-β1/β2(94-98) inhibited the growth of LS513 cells with an ED₅₀ of approximately 1000 pM. TGF-β1/β2(95-98), in which amino acids 95-98 of TGF-β1 have been replaced with the corresponding amino acids of TGF-β2, had full activity and was indistinguishable from TGF-β1. 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-β1/β2(92-98), TGF-β1/β2(92-95) and TGF-β1/β2(94-98) were each similar to TGF-β2 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-β and its signaling receptors and they show that modest changes within this region can substantially alter biological response.

Key Words:

• Colon cancer
• protein structure
• growth inhibition
• isoform