Figure 1.

The Lrp6-APEX2 fusion protein receptor retains signal transducing activity, proper subcellular localization, and biotinylates cellular proteins. (A) Schematic diagram of the Lrp6-APEX2 construct. The human Lrp6 contains an N-terminal VSV-G and a linker Flag-tag followed by the APEX2 enzyme. The construct also contains an internal ribosomal entry site (IRES) followed by a GFP-tagged Puromycin N-acetyltransferase (PAC) used for selection of permanently transfected cells. (B) Diagram depicting the biotinylation of Lrp6-APEX2 proximal targets. During the Wnt signal, in the presence of Biotin-phenol (BP) and H₂O₂, proteins recruited to the receptor are biotinylated by the APEX2 peroxidase, including known Wnt targets (Dvl, Axin, APC, etc.) and novel targets (X, Y). (C–E) control embryos from oocyte-host transfer experiments show normal development (100%, n = 24), as compared to oocytes depleted with a phosphorothioate antisense DNA oligo targeting Xenopus Lrp6²⁷, which develop as ventralized embryos (75%, n = 22). Co-injection of 300 pg of Lrp6-APEX2 mRNA into oocytes completely rescued axis formation (60%, n = 17), indicating that the fusion protein is fully active. Scale bar represents 500 µm. (F–H) Lrp6-APEX2 is trafficked to the plasma membrane in stably transfected HEK293T cells. Lrp6 was detected through its Flag-tag and Phalloidin, which stains cortical F-Actin, confirmed cell surface localization of Lrp6. Scale bar represents 10 µm. (I–L) Lrp6-APEX2 changes subcellular localization by Wnt, Dkk1 and Bighead treatments. In presence of control conditioned medium (CM), Lrp6 is located mostly at the plasma membrane (79%). Treatment with Wnt3a CM for 30 min increases the number of intracellular vesicles containing Lrp6 (53%). Treatment with Dkk1 protein (200 ng/ml) or Bighead CM induced relocation of Lrp6 to the juxtanuclear bay area where lysosomes are located (63% and 53%, respectively). Quantification was obtained counting cells showing the observed immunostaining over total nuclei, in 10 different micrographs. Scale bars represent 10 µm. (M) western blot stained with Streptavidin-IRDye 800 (in green) showing controls indicating that Lrp6-APEX2 biotinylates proximal proteins only in the presence of both BP and H₂O₂ (compare lane 5 with 2–4). Negative control cells not expressing the APEX2 peroxidase had no biotinylation even in presence of BP/H₂O₂ (lane 1). Note the presence of three Streptavidin-positive bands in all lanes, which correspond to endogenous biotinylated carboxylases. Gapdh served as a loading control.