Comparison of LRP5 and LRP6 Figure 1. Lrp62/2 MEFs are less responsive than Lrp52/2 or WT MEFs to Wnt3a stimulation. (A) Endogenous expression of LRP5 and LRP6 in the mutant MEFS from total cell lysates. b-tubulin was used as a loading control. (B) Accumulation of cytoplasmic b-catenin in response to 6 hours of treatment of control L cell conditioned media (L) or L-Wnt3a conditioned media (W3A)examined via immunoblotting. Densitometry of cytosolic b-catenin levels, normalized to cytsolic b-tubulin loading control bands, are shown below each lane. doi:10.1371/journal.pone.0023537.g001 contained the C, D and E motifs of LRP6).LRP6/5CDE (which contained the A and B motifs of LRP6).LRP6/5C (Figure 3C and 3D). We found similar results when LRP5 replaced *LRP5 in the generation of analogous chimeric receptors, and with either Wnt1 or Wnt3a (Figure S2A and S2B, and data not shown). These results together suggest that the first half (containing the A and B motifs) and the second half (containing the C, D, and E motifs) of LRP6 each confer more signaling activity than their LRP5 counterparts, and that the second half of LRP6C confers the strongest signaling activity. These results are consistent with our previous studies that LRP6 signaling is mostly mediated by C plus D plus E motifs [28,29]. Comparison of Axin interaction with phosphorylated LRP5 and LRP6 Axin recruitment to activated/phosphorylated LRP5 or LRP6 has been well documented [30,31,32] and is believed to be a key step in Wnt signal transduction [3]. The significant difference between the Wnt signaling activity of LRP5C and LRP6C is surprising considering that the five PPPSPxS motifs, which are phosphorylation-dependent Axin docking sites, are virtually iden- tical pair-wise between LRP5 and LRP6 (Figure S1B). We attempted to compare Axin binding to LRP6 and LRP6/5C, which exhibited highest and lowest signaling activities, respectively, via coimmunoprecipitation in cells. However as activated LRP6 signaling degrades the Axin protein as previously observed [33], we found that LRP6 expression resulted in much lower Axin levels compared to LRP6/5C expression (at both one and two days post- transfection, data not shown). Since these unequal Axin levels complicated the binding experiment, we chose to use a different in vitro approach that examines binding between Axin and recombi- nant LRP5C or LRP6C (fused with GST, glutathione S transferase). Previously we used this method to show that GST-LRP6C, upon phosphorylation by GSK3 plus CK1 in vitro, binds to Axin [13]. We purified GST-LRP5C (last 207 a.a. of LRP5) and GST-LRP6C (last 218 a.a. of LRP6), and subjected them to in vitro phosphorylation with recombinant GSK3 plus CKI in the presence (or absence) of ATP. Similar to phosphorylated GST-LRP6C [13], phosphorylated GST-LRP5C exhibited a slower migration pattern in gel electrophoresis (Figure 4A). Given the pair-wise conservation between PPPSPxS motifs of LRP5 and LRP6, we used phospho- specific antibodies that we have raised against motifs A, C, and E of LRP6 to examine the relative amount of phosphorylation of GST- LRP5C and GST-LRP6C. We detected similar intensities of phosphorylation at these three motifs with Ab1490 for phospho- motif A [12], Ab1572 for phospho-motif C [28], and Ab1607 for phospho-motif E [28] (Figure 4B–D). These antibodies by large were specific for phosphorylation of both LRP5 and LRP6 (Figure 4 B–D), although a minor exception was Ab1490, which cross-reacted slightly with unphosphorylated GST-LRP5C (Figure 4B). None- theless these experiments showed relatively comparable levels of phosphorylation of LRP5C and LRP6C by GSK3 and CK1 in vitro. Importantly, we found that phosphorylated, but not unpho- sphorylated, LRP5C and LRP6C each bound to the Axin protein from cell lysates at apparently comparable levels (lanes 6 and 9, Figure 4G). These data suggest that LRP5 and LRP6, if phosphorylated similarly, exhibit similar abilities to bind/recruit Axin. Axin-LRP5/6 association is unlikely bridged by GSK3 How phosphorylated/activated LRP5 or LRP6 binds to Axin remains somewhat debated. The initial finding that Axin binds to LRP5C in the yeast two-hydrid assay implied that the interaction might be direct [30], and phosphorylated PPPSPxS motifs in LRP6C were subsequently shown to mediate Axin-docking [12,13], supporting a LRP5/6-Axin direct binding model. However since LRP6 can also bind to GSK3 [13,34], possibly through PPPSP motifs that are substrates for GSK3 [13,35], and/ or through an upstream serine/threonine-rich region in LRP6 (Figure S1B) [36,37], an alternative model was proposed that GSK3, which is known to complex with Axin, acts as a bridge between LRP6 and Axin [36]. We therefore designed experiments to test these two models for both LRP5 and LRP6. After phosphorylation of GST-LRP5C and GST-LRP6C by GSK3 plus CK1 we extensively washed these GST-fusion proteins on glutathione beads using a high salt condition (Figure 4E). Compared to input GSK3 and GSK3 reference standards, GST-LRP5C and GST-LRP6C proteins post washing retained a small amount of GSK3, which was independent of LRP5C/ LRP6C phosphorylation and was not found in the lane of GST alone (Figure 4E), consistent with the direct and specific LRP6- GSK3 binding as previously reported [13,34,35]. However these LRP5/6-bound GSK3 proteins were not capable of recruitment of Axin into the complex (lanes 5 and 8, Figure 4G). The serine/ threonine cluster upstream of PPPSPxS motifs was suggested to be a GSK3-binding site that can mediate LRP6C-GSK3-Axin interaction [36]. We generated a 14-amino acid deletion of this S/T cluster, LRP6CDS/T (Figure S1B), which we found exhibited phosphorylation-dependent Axin binding that was indistinguish- able from LRP6C (Figure 4H). Our results therefore do not support the model that GSK3 bridges LRP5/6-Axin association and are consistent with a direct LRP5/6-Axin binding through phopshorylated PPPSPxS motifs. PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23537