LGR4 is required for RSPO1-induced Wnt/β-catenin signaling.

<p>(a) Depletion of LGR4 inhibits RSPO1-, but not Wnt3a-, induced STF reporter activation. HEK293T-STF cells were transfected with pGL2, LGR4, LGR5, or LRP6 siRNA’s. 40 h after transfection, cells were treated with RSPO1 or Wnt3a-conditioned medium overnight, and luciferase was measured. pGL2 siRNA was used as negative control. (b) Expression of LGR4, LGR5 and LGR6 rescue LGR4 siRNA-elicited decrease in RSPO1-induced STF reporter activation. HEK293T cells stably expressing siRNA-resistant LGR4 (LGR4-R), LGR5, LGR6, LGR2 or vector control were transfected with pGL2 siRNA or LGR4 siRNA, and corresponding STF values were determined. Expression of LGR4-R, LGR5 and LGR6, but not LGR2, could rescue LGR4 loss. Luciferase values were normalized relative to pGL2 siRNA. (c) LGR4 siRNA specifically reduces RSPO1-induced LRP6 phosphorylation and β-catenin stabilization. HEK293T cells transfected with indicated siRNA’s were treated with RSPO1 or Wnt3a-conditioned medium overnight. Phospho-LRP6, cytosolic β-catenin and tubulin were analyzed by immunoblotting using indicated antibodies.