Figure 5.

Biological activities of phenotypically relevant GDF-5 variants. (A) Induction of ALP expression in RobC26 cells. GDF-5 exhibits an EC₅₀ of about 10 nM, whereas R57L requires a 10-fold lower concentration for half-maximal stimulation. All other variants showed no ALP induction in RobC26 cells. (B) As in (A), but using ATDC5 cells that lack BMPR-IB receptor. The variant GDF-5R57L exhibits an ∼2-fold lower EC₅₀ value correlating nicely with the two-fold increased affinity for BMPR-IA. The variant S58T, which is unable to induce ALP expression in RobC26 cells shows a residual activity in ATDC5 cells. (C) Reporter gene activation in C3H10T1/2 cells stably transfected with a p(BRE₂)-Luc reporter construct. GDF-5 and its variant R57L with an increased affinity for BMPR-IA show a dose-dependent activation of the SMAD1/5/8 pathway. As in the ALP expression studies, all other variants that have either a decreased type I receptor affinity or exhibit an accelerated complex dissociation rate are inactive in SMAD activation. (D) As in (C), but using ATDC5 cells that were transiently transfected with the p(BRE₂)-Luc reporter construct.