Fig. 8. Wise interacts with the extracellular domain of LRP6. (A-C) All components were provided as concentrated conditioned medium for the immunoprecipitation study. Asterisks show samples of input loaded directly on to the gel. Extracellular domains of LRP6 and Frizzled8 are fused to human IgG Fc domain. E1-2 and E3-4 are deletion constructs of LRP6 in which the first two or last two EGF repeats respectively are missing (Mao et al., 2001). (A) Wise binds to LRP6 and a deletion construct of LRP6 (E3-4), but not to Frizzled8 or LRP6 E1-2. Dkk1 binds to LRP6 and E1-2 LRP6, but not to E3-4 LRP6 or Frizzled8. Wnt8 binds to LRP6 and Frizzled8. (B) Wise does not bind to Wnt8. Dkk1 and extracellular domain of Frizzled1 (Fz1 ECD) are used as a negative and a positive control for binding to Wnt8, respectively. (C) Binding of Wnt8 and LRP6 is attenuated in the presence of Wise, but binding of Wnt8 and Frizzled8 is not. (D) A possible model of Wise action on Wnt signalling. Open arrows in each panel show weak (1,3) and strong (2) activation of the downstream pathway. (1,2) Wise and Wnt share the same binding domain of LRP6, while Dkk1 interacts with a different domain on LRP6. Wise induction of downstream targets of the canonical Wnt pathway is weaker compared with Wnt. (3) Wise competes with Wnt for binding to LRP6, resulting in attenuation of the Wnt action (compare 2 with 3).

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