FIGURE 4.

Ectopic expression of either LRP5 or LRP6 eliminates requirement for both LRP5 and LRP6. A, overexpression of LRP5 and LRP6 in wild type MEFs. Wild type MEFs were transfected with constructs encoding full-length mLRP5, mLRP6, or a control construct (GFP plasmid; Mock). 36 h later, protein extracts were probed with anti-LRP antibodies as indicated, and the relative amount of expression was quantified. B–D, assay of ligand dependence of signaling in MEFs overexpressing LRP species. To test whether MEFs overexpressing LRP receptors to the levels reported in A were still ligand-dependent for Wnt transactivation, cells were transfected with the TOP-FLASH Wnt reporter (B), and luciferase activity was measured 36 h later. Signaling was undetectable (over background) without the introduction of Wnt ligands (D). To test the signaling properties of single receptor species in knock-out backgrounds, wild type, lrp5−/−, and lrp6−/− MEFs were transfected with the Wnt reporter superTOP-FLASH (and the transfection control Renilla luciferase) together with mWnt3a (B) or mWnt1 (C) and either LRP5 or LRP6 receptor. 36 h post-transfection, cells were lysed, and Wnt signaling activity was measured. UT, untransduced; RLU, relative luminescence units. Error bars show standard deviations.