Comparison of LRP5 and LRP6 Figure 6. The context and spacing of the gap4 region together reciprocally regulate LRP5 and LRP6 signaling activities. (A and B) Additional derivatives in the gap4 region (underlined) of *LRP5 and LRP6. The mutated residue of each derivative is in bold. Motifs D and E are boxed and known sites phosphorylated by GSK3 (#) and CKI (*) are indicated below the alignment. The LRP6 gap4 serine is not predicted to be a CKI phosphorylation site. (C–D) TOPFLASH reporter activity of *LRP5 and derivatives alone (C) or co-transfected with Wnt1 (D). (E–F) TOPFLASH reporter activity of LRP6 and derivatives alone (E) or co-transfected with Wnt1 (F). The LRP6e mutant [28], which has PPPAP instead of PPPSP in motif E, was used for comparison. doi:10.1371/journal.pone.0023537.g006 gap4 exhibits stronger signaling comparable to LRP6, whereas LRP6 with an altered and ‘‘LRP5-like’’ gap4 region exhibits weaker signaling similar to LRP5 (Figures 5 and 6). Indeed our data suggest that the contribution by gap4 to LRP6 signaling is on par to that by motif E and is more prominent than that by motif A or B (Figure 6) [28,29]. It has been established that Axin binds to both LRP5 [30] and LRP6 [32], and that phosphorylated PPPSPxS motifs provide Axin-docking sites that mediate LRP6 signaling [12]. The difference between LRP5 and LRP6 signaling could therefore be a result of their different Axin-binding properties. But we found that recombinant LRP5C and LRP6C, upon in vitro phosphor- ylation by GSK3 and CK1, exhibit indistinguishable phosphor- ylation-dependent binding to Axin (Figure 4), suggesting that the difference of LRP5 and LRP6 signaling in vivo is likely at a step prior to Axin-binding, i.e., at how effectively the PPPSPxS motifs are phosphorylated. Fully consistent with this possibility, we found that, using antibodies specific for phospho-A, -C, and –E, LRP6 is more readily phosphorylated at these PPPSPxS motifs than its LRP5 counterparts (Figure 5). More revealingly, the gap4 region in LRP5 when altered to resemble that in LRP6 not only enhances LRP5 signaling but also LRP5 phosphorylation at these PPPSPxS motifs (Figure 5C). On the other hand other gap regions (gaps1, 2, and 3) have minimal effects on LRP5 signaling or phosphorylation (Figure 5). Thus we suggest that the difference in LRP5 and LRP6 signaling activity is largely due to their effectiveness of phosphor- ylation at PPPSPxS motifs, and we have identified the gap4 region that is responsible, at least in a significant part, for the difference between LRP5 and LRP6 phosphorylation and signaling. The gap4 region lies between motifs D and E, but it has a strong effect on phosphorylation of not only motif E (and possibly D), but also of motifs C and A that are some distance away (Figure 5C and Figure S1B). We believe that this is consistent with the ‘‘LRP6 signal amplification’’ model we previously proposed [28], which corroborates genetic observations in Drosophila embryos [39]. This model is based on the observation of a local positive feed forward loop between Axin and LRP6 PPPSPxS motifs. Thus Axin not only binds to phosphorylated PPPSPxS motifs, but also promotes and is required for PPPSPxS phosphorylation via its recruitment of the Axin-GSK3 complex [28,40]. Therefore phosphorylation of one PPPSPxS motif has a stimulatory effect on that of other PPPSPxS motifs. Indeed we previously demonstrated that LRP6 phosphorylation at C or E profoundly relies on the presence of other PPPSPxS motifs [28]. Here we interpret that the gap4 region, by directly regulating phosphorylation at motif E (and possibly motif D) nearby, exerts significant effects on phosphor- ylation of other motifs such as A and C (Figure 5) through such a signal amplification mechanism. This may also explain why gap4 has a prominent role in the overall LRP5 and LRP6 signaling output. PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23537