The gap4 sequence SYF is conserved among vertebrate Lrp5 proteins, and is distinct from SYSH in gap4 that is invariable among vertebrate Lrp6 orthologs (Figure S1B). Such a conserved sequence difference together with its critical modulation of LRP5 and LRP6 signaling activity are unlikely to be co-incidental. Although the serine and tyrosine residues in gap4 call for potential (and differential) phosphorylation regulation in LRP5 and LRP6, our mutational analyses do not seem to support such a scenario (Figure 6 and data not shown). How does gap4 regulate phosphorylation at PPPSPxS motifs and thereby LRP5/LRP6 signaling remains to be investigated. One possibility is that gap4 in LRP5 or LRP6, perhaps in conjunction with flanking residues, serves as a binding site for an unknown protein. Alternatively the sequence difference between LRP5 and LRP6 results in conformational difference that affects relative positioning of the last two PPPSPxS motifs (D and E), impacting signal amplification. Intriguingly the predicted secondary structure of LRP6 gap4 region contains a ‘‘turn’’ that is lacking in LRP5, and furthermore. the LRP5+gap4S mutant gains, while the LRP6 SH.F mutant loses, this predicted turn (Figure S3), correlating with the increase and decrease of receptor signaling strength, respectively. We note that while other gap regions, including gap2, do not appear to influence LRP5/6 activities in Wnt/b-catenin signaling, a recent study has shown that the RMTSV region of LRP6 gap2 serves as a potential phosphorylation site (T1558) for Protein Kinase A (PKA) and mediates LRP6 and Gas interaction in response to parathyroid hormone (PTH) binding to LRP6 [41]. This PTH responsive PKA site in gap2 is absent in Lrp5 (Figure S1B). Although our data do not favor a similar phosphorylation- dependent regulation of gap4, this and our studies together highlight important regulatory roles of different gap regions between PPPSPxS motifs in LRP5 and LRP6 in Wnt and possibly other signaling pathways. Our study further helps to resolve a controversial issue regarding LRP5/6-Axin interaction. Earlier findings based on yeast two-hybrid assays suggested that LRP5/6-Axin association is likely direct [30] and is mediated through phosphorylated PPPSPxS motifs as Axin-docking sites [12,13], although this model has not ruled out the caveat that GSK3-like proteins in yeast may have a role in mediating the two-hydrid interaction. This direct interaction model is complicated/challenged by the findings that GSK3 also binds to LRP6 and performs PPPSP phosphorylation [13,34,36,37], and that phosphorylated PPPSPxS peptides can inhibit GSK3 phosphorylation of b-catenin presum- ably through direct interaction with GSK3 [35,36]. Furthermore the serine/threonine-rich region upstream of the PPPSPxS motif A (Figure S1B) may also bind to GSK3 [36,37]. Given these scenarios and the established Axin-GSK3 interaction, a recent in vitro study has argued that Axin-LRP6 interaction is indirect and requires GSK3 as an intermediate bridge [36]. Using recombinant GST-LRP5C or -LRP6C plus GSK3 (and CK1) we performed in vitro reconstitution of phosphorylation-dependent LRP5C/ LRP6C-Axin interaction. We found that GSK3 indeed binds to GST-LRP5C and -LRP6C, but not to control GST, this binding is however independent of LRP5C/LRP6C phosphorylation (Figure 4). By contrast, Axin binds only to GST-LRP5C and - LRP6C that have been phosphorylated by GSK3 plus CK1 (Figure 4). Importantly the presence of GSK3 with LRP5C/ LRP6C does not result in Axin recruitment (Figure 4G). Furthermore, LRP6CDS/T, which harbors a deletion of the serine/threonine-rich region that was suggested to be a GSK3- binding site [36,37], binds to Axin in a phosphorylation-dependent manner that is indistinguishable to the wild type LRP6C (Figure 4H). Therefore our results are consistent with a direct Comparison of LRP5 and LRP6 LRP5/6-Axin interaction and do not support the model that GSK3 is the intermediate bridge between LRP5/6 and Axin. We note that in experiments supporting the GSK3-bridging model [36], the authors employed an Axin fragment that lacks the so- called DIX domain, which was suggested to be required for LRP5/6-Axin interaction [30]. In summary we have determined that the cytoplasmic domain of LRP5 and LRP6 plays a major role in the different signaling activity of these two Wnt receptors, and identified between the last two carboxyl PPPSPxS motifs an intervening gap4 region that has a key modulatory function in LRP5/LRP6 phosphorylation and signaling output. We have also provided evidence that argues for direct LRP5/LRP6-Axin interaction. Collectively our data provide significant new insights into the molecular mechanism of LRP5/LRP6 in Wnt signal transduction in development and human diseases. Materials and Methods Plasmids Human LRP5 and LRP6 were tagged with the VSVG epitope and cloned into pCS2+ as previously described [12]. When not present in the endogenous sequence, unique restriction enzyme sites were introduced using the QuickChange Mutagenesis Kit (Stratagene). Point mutants and c-terminal insertion/deletion mutations were created in the LRP5DN and LRP6DN constructs, and later transferred over to the full-length receptor using the ClaI site before the transmembrane domain. LNCX-Wnt1, LNCX- Wnt3a or an empty LNCX vector, was used for co-expression assays. GST-LRP5C (amino acids 1409–1615) was cloned into pGEX4 using similar boundaries based on GST-LRP6C (amino acids 1396–1613). GST-LRP6CDS/T contains a 14 amino acid deletion (D1466–1479, see Figure S1B). For experiments analyzing full-length LRP5/6 phosphorylation, pCS2-MESD was added at a 10:1 LRP5/6:MESD ratio to facilitate processing and trafficking of the receptors to the membrane. Full details of all plasmids are available upon request. Dual luciferase assay Mammalian cell transfections were done in HEK 293T cells (ATCC# CRL-11268) using FuGENE 6 and performed in triplicate. Cells were plated at 16105/ml in 24 well plates and transfected the following day with a total of 300 ng of DNA/well [50 ng TOPFLASH, 10 ng TK-Renilla, 90 ng pCS2+ (empty vector), 50 ng of LNCX/Wnt1 and 100 ng of LRP5/6 constructs or pCS2 vector control]. Dual luciferase reporter assays were performed as previously described [28]. Representative results are shown from one of three (or more) independent experiments. In vitro phosphorylation and binding assays Recombinant GSK3 (GSK3b: P6040, NEB) and CKI (CKId: P6030, NEB) were used for in vitro phosphorylation and binding assays as previously described [13]. Following the in vitro phosphorylation reaction with 250 units of GSK3 and 500 units of CKI, GST proteins were thoroughly washed to minimize the amount of recombinant GSK3 and CKI in the future binding assay (46 using a high salt condition: 50 mM HEPES pH 7.4, 500 mM NaCl, 1.5 mM EDTA, 1% Triton X-100, 10% glycerol). The same HEPES buffer containing only 150 mM NaCl was used for Axin binding conditions and post-binding washes (66). Immunoblotting and antibodies Polyclonal anti-VSVG (V4888, Sigma) was used to detect the expressed LRP5/6 chimeric receptors by western blot analysis as PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23537