Comparison of LRP5 and LRP6 Figure 2. LRP5 and LRP6 cytoplasmic domains (LRP5C and LRP6C) show significant difference in signaling activities. (A) Schematic representation of LRP5/LRP6. SP: signal peptide; 1–4, propeller plus EGF-repeat 1 to 4; LDLRA: LDL receptor type A repeats; TM: transmembrane domain; A–E: PPPSPxS motifs A to E. BspEI was introduced together with the VSVG tag after the SP (Figure S1A); ClaI was introduced immediately after the TM domain and the endogenous PstI is immediately after the motif B (Figure S1B). These restriction sites were used to generate chimeric receptors. (B) Schematic LRP5 (blue) and LRP6 (red) used for the signal peptide exchange experiment. (C) LRP5, *LRP5, and LRP6 levels in lysates from HEK293T cells that were transfected with the respective expression plasmid, as detected by a VSVG antibody. (D and E) TOPFLASH reporter activity of LRP5, *LRP5, and LRP6 alone (D) or co-expressed with Wnt1 (E) in HEK 293T cells. Wnt responsive TOPFLASH (firefly) luciferase units were internally controlled to the non-Wnt responsive Renilla reporter, and were normalized to vector control and presented as relative luciferase units (RLU). The dotted green line in E represents the activity of Wnt1 alone (through the endogenous receptor). (F) Schematic LRP5 (blue) and LRP6 (red) used for cytoplasmic domain swap experiments. (G and H) TOPFLASH reporter activity of chimeric LRP5/6 receptors expressed alone (G) or co-expressed with Wnt1 (H). doi:10.1371/journal.pone.0023537.g002 Amino acid residues between PPPSPxS motifs D and E modulate LRP5/6 signaling activity Our in vitro binding results suggested that when similarly phosphorylated, LRP5 is as effective as LRP6 in binding to Axin, consistent with the high conservation of the five PPPSPxS motifs found in LRP5 and LRP6. However in the context of the full- length receptor, LPR5C is much less effective in activating Wnt signaling. One possible explanation is that LRP6 is more readily phosphorylated than LRP5 in vivo, and the amino acid residues outside the conserved PPPSPxS motifs may be responsible for the observed functional difference. An alignment of LRP5C and LRP6C from multiple vertebrate species reveals several gaps, PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23537