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. 2003 Aug;23(16):5825–5835. doi: 10.1128/MCB.23.16.5825-5835.2003

FIG. 6.

FIG. 6.

Wnt ligand interactions with the oligomeric LRP6 external domain relieve allosteric inhibition on the LRP6 intracellular domain. (A) Constitutive tyrosine autophosphorylation of a LRP6-FGFR chimeric receptor. Flag-LRP6-FGFR (1 μg) and Flag-FGFR2 (0.5 μg) were transfected in 293T cells. Basic FGF (bFGF) (100 ng/ml) was added at 24 h to Flag-FGFR2-transfected cells. At 48 h, lysates (2 mg) were immunoprecipitated (IP) with anti-Flag antibody and were subjected to SDS-PAGE analysis and immunoblotting (IB) with anti-p-Tyr polyclonal antibody. The same blot was stripped and reprobed with anti-Flag antibody to determine receptor protein levels. (B) Complex formation between Wnt ligand and LRP6 receptors. 293T cells were transfected with 1 μg of Wnt3a-HA together with 1 μg of Flag-FGFR2, Flag-GyrB-LRP6, Flag-LRP6, or Flag-LRP6-FGFR. At 48 h, cell lysates were immunoprecipitated with anti-HA antibody followed by SDS-PAGE and immunoblot with anti-Flag or anti-HA antibody. (C) Wnt3a but not Dkk-1 inhibits constitutive tyrosine autophosphorylation of the LRP6-FGFR chimera. 293T cells were transfected with Flag-LRP6-FGFR (1 μg) and either vector (Vec), Dkk-1-HA (3 μg), or Wnt3a-HA (3 μg). At 48 h, cell lysates were immunoprecipitated with anti-Flag antibody followed by SDS-PAGE and immunoblotting with either anti-p-Tyr, anti-Flag, or anti-HA antibody. Normalized quantification of the immunoblots is shown at the bottom. (D) Activation of LRP6 receptor and inhibition of constitutive receptor tyrosine autophosphorylation of LRP6-FGFR by exogenously added Wnt3a CM. Twenty-four hours following transfection of 293T cells with 0.5 μg of vector or LRP6, control (Con) CM (from L cells, ATCC no. CRL-2648) or Wnt3a CM (prepared from Wnt3a stably transfected L cells, ATCC no. CRL-2647) was added for 1 h, and the cell lysates were subjected to uncomplexed β-catenin analysis (upper panel). Twenty-four hours following transfection of 0.5 μg of Flag-LRP6-FGFR in 293T cells, control CM or Wnt3a CM was added for various times as indicated, and cell lysates were then immunoprecipitated with anti-Flag antibody followed by SDS-PAGE and immunoblot analysis with anti-p-Tyr. The same blot was stripped and reblotted with anti-Flag (lower panel). (E) Wnt ligand induces conformational alteration in LRP6 receptor oligomers. 293T cells were transfected with 1 μg of Flag-LRP6-FGFR and myc-LRP6-FGFR together with 4 μg of either vector or Wnt-1-HA plasmids. At 48 h, cell lysates were immunoprecipitated with anti-Flag antibody followed by SDS-PAGE and immunoblotting with anti-myc, anti-p-Tyr, anti-Flag, or anti-HA antibody as described above. (F) Effects of Wnt-1 and Dkk-1 on oligomerization of the wild-type LRP6 receptor. 293T cells were transfected with 0.5 μg of Flag-LRP6-FGFR and myc-LRP6-FGFR together with 2 μg of vector, Wnt-1-HA, or Dkk-1-HA plasmid. At 48 h cell lysates were immunoprecipitated with anti-Flag antibody followed by SDS-PAGE and immunoblotting as described above.