Comparison of LRP5 and LRP6 Figure 3. Additional analyses of the cytoplasmic domain of LRP5 and LRP6. (A) Schematic LRP5 (blue) and LRP6 (red) receptors used for the cytoplasmic domain swap experiment. (B) Chimeric LRP5/6 protein levels in lysates from HEK293T cells transfected with the respective expression plasmid, as detected by a VSVG antibody. (C and D) TOPFLASH reporter activity of chimeric LRP5/6 transfected alone (C) or co-transfected with Wnt1 (D). doi:10.1371/journal.pone.0023537.g003 referred to here as gaps1–4, where LRP6 contains additional amino acid residues between PPPSPxS motifs (Figure S1B). We first introduced these gap amino acid residues into LRP6/5C, which behaved inactive or slightly dominant negative. Addition of Gap1, Gap 2 or Gap3 did not significantly alter the activity of LRP6/5C (Figure 5A). To our surprise, the addition of the gap4 serine residue between motifs D and E in the LRP6/5C+gap4 mutant resulted in high signaling activity that was comparable to LRP6 (Figure 5A). LRP6/5C+gap4 also resembled LRP6 when co-expressed with Wnt1, while LRP6/5C or each of LRP6/ 5C+gap1/2/3 derivatives behaved inactive (in fact dominant negative) (Figure 5B). Importantly, we detected phosphorylation at motifs A, C, and E in LRP6 and LRP6/5C+gap4, but not in LRP6/5C or LRP6/5C+gap1/2/3 (Figure 5C). These data are consistent with the idea that LRP5C is less active in vivo due to poorer phosphorylation, and demonstrate that introduction of gap4 is sufficient, at least in part, to enhance LRP5 phosphory- lation and signaling. We next introduced these gaps in the full length *LRP5. *LRP5+gap4, but none of the *LRP5+gap1/2/3 derivatives, resulted in elevated signaling activity with Wnt1 to a level comparable to that of LRP6 (Figure 5D). The serine residue in the gap4 region was intriguing and hinted the possibility of regulation via post-translational modifications such as phosphorylation. However this seemed unlikely as an alanine insertion in the place of the serine residue still conferred significant signaling enhancement in the *LRP5+gap4A mutant (Figure 6D). Further inspection of the gap4 region in LRP5, SYF, versus SYSH in LRP6 (Figure 6A and 6B), prompted us to generate *LRP5F.H, in which the phenylalanine (LRP5 residue 1601) was replaced by histidine. *LRP5F.H also exhibited significant higher activity than *LRP5 (Figure 6D). We thus considered the scenario that the tyrosine residue and its possible phosphorylation in gap4 may be differentially regulated in LRP5 versus LRP6 due to the neighboring F versus H change (Figure 6A, 6B and Figure S1B). But this also seemed unlikely because replacement of the tyrosine by phenylalanine had little effect on signaling by either *LRP5 or LRP6 (data not shown). These results suggest that the collective context of gap4 residues plays a major role in the activity difference between LRP5 and LRP6. We generated reciprocal changes in LRP6. Removing gap1 or 2 or 3 in LRP6 did not affect its signaling function (data not shown), mirroring the little effect of insertion of these gaps each in LRP5 (Figure 5A and 5B). Similar to the *LRP5 data that the serine residue per se in gap4 is not essential, a deletion of serine 1598 or its replacement by alanine in LRP6DS1598 and LRP6S1598A, respectively, did not affect LRP6 signaling (Figure 6E and 6F). Importantly however, a substitution of serine 1598 and histidine 1599 in LRP6 gap4 with phenylalanine, generating the LRP6SH.F mutant that had a gap4 resembling that in LRP5, significantly diminished LRP6 signaling activity (Figure 6E and 6F). Note that the reduction of activity in LRP6SH.F was on par to that observed in the LRP6e mutant, which has a non-functional PPPAP alteration in motif E, and was much more pronounced than that observed in LRP6a or LRP6b mutants harboring a non- functional PPPAP alteration in motif A and B, respectively (Figure 6E and 6F) [28]. Thus the gap4 contribution to LRP6 signaling is similar to that by PPPSPxS motif E but more significant than that by motif A or B. These reciprocal data in LRP5 and LRP6 demonstrate that the gap4 region between the last two PPPSPxS motifs (D and E) accounts for most of the PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23537