inhibitory function of the antibodies is dependent on blocking Wnt binding to LRP6 (Figure 3D). It is not clear why the activity of the Wnt-LRP6 chimeras are also largely insensitive to potentiation of Wnt signaling by the antibodies, nor why the Wnt3a-LRP6 fusion is insensitive to Fzd8CRD-Fc inhibition. The lower level of signaling induced by LRP6 overexpression is weakly affected by YW211.31.57 and YW210.09 antibodies, suggesting that a normally undetectable level of endogenous Wnt signaling in HEK293 cells has been amplified. Antibody-mediated Potentiation of Wnt Signaling Involves LRP6 Dimerization Potentiation of autocrine Wnt signaling by YW211.31 antibody requires avidity effects, presumably through LRP6 dimerization. A monovalent recombinant one-armed YW211.31.62 antibody (Figure 4A–4C) and the Fab fragment of YW211.31.62 (data not shown) exhibit no potentiation of autocrine Wnt signaling in EKVX and Hs578T cells at concentrations that inhibit Wnt3a- induced signaling in these cell lines. In contrast, the one-armed YW211.31 antibody and Fab fragment inhibit both autocrine Wnt and Wnt3a-induced signaling in the PA-1 cell line with similar potency to the intact IgG antibody (Figure 4D). As expected, the YW211.31.57 whole antibody, but not the one-armed antibody, is able to induce aggregation of differentially tagged LRP6 proteins at the surface of HEK293 cells (Figure S3). To test whether crosslinking of the one-armed YW211.31 antibody would restore the Wnt potentiation function of the whole IgG molecule, we used the HT-1080 cell line that exhibits autocrine Wnt and Wnt2-induced signaling potentiated by YW211.31.57 antibody (Figure 4E and 4F). The one-armed YW211.31 antibody has no effect on autocrine Wnt signaling or signaling induced by Wnt2 transfection. HT-1080 cells also display Apomab antibody- induced apoptosis that is enhanced by Fc crosslinking (Figure S4) [26]. Under crosslinking conditions with anti-Fc antibodies that augment Apomab-mediated apoptosis, we find that crosslinking of the one-armed antibody partially reconstitutes the potentiation of both autocrine Wnt and Wnt2 signaling observed with the YW211.31.57 whole antibody (Figure 4E and 4F). Inhibiting the activity of extracellular LRP6 antagonists such as DKK1 can also potentiate Wnt signaling [27]. Exogenous DKK1 protein partially inhibits Wnt1-induced signaling in HEK293 cells, and YW211.31.57 antibody can neutralize this antagonism and even weakly potentiate signaling at high enough concentration in the presence of DKK1 protein (Figure 4G). In contrast, one-armed YW211.31 antibody only very weakly and partially counteracts DKK1 antagonism of Wnt1 signaling at these same concentra- tions. YW211.31.57 whole antibody effectively antagonizes DKK1 activity at all DKK1 concentrations tested, whereas the one-armed antibody has minimal or no effect even at low DKK1 concentration. While we have not demonstrated that that endogenous DKK1 is present and active in HEK293 cells, the potent antagonism of exogenous DKK1 activity observed with the whole but not the one-armed YW211.31 antibody may contribute to the Wnt-potentiating activity specific to the whole antibody. Alternatively, since DKK1 protein was not able to inhibit completely the Wnt1-induced signaling in this assay, it is also possible that the intact YW211.31 antibody simply potentiates the remaining signal through LRP6 dimerization. Wnt Signaling Antagonism Predominates in LRP6 Antibody Combinations Collectively, our results suggest that Wnt3a and Wnt3 bind within the E3-E4 region of LRP6 and are inhibited from binding Wnts Specify LRP6 mAb Activity by YW211.31.57 antibody. Wnt isoforms in the Wnt1 class are predicted to bind the E1-E2 region, and this binding may be blocked by YW210.09 antibody. In this model, potentiation of Wnt signaling would occur when both a Wnt isoform and an antibody are able to bind the same LRP6 molecule, presumably requiring recruitment of FZD by the Wnt and LRP6 dimerization by the antibody. This model predicts that the combination of both antibodies would inhibit signaling induced by either class of Wnt isoforms since, although LRP6 dimerization would likely still occur, Wnt binding would be blocked by one or the other antibody. As predicted, treating HEK293 cells simultaneously with YW211.31.57 and YW210.09 antibodies inhibits signaling initiat- ed by expression of either Wnt3a or Wnt1 (Figure 5A). We extended this analysis to three other Wnt isoforms in the Wnt1 class and found that the combination of YW211.31.57 and YW210.09 antibodies inhibits Wnt signaling to a similar extent as YW210.09 alone (Figure 3E). When both Wnt3a and Wnt1 are expressed simultaneously, neither antibody antagonizes Wnt signaling, but the combination of both antibodies does inhibit signaling (Figure 5A). This result can be explained if each antibody inhibits binding of only one Wnt isoform, and both antibodies are able to bind LRP6 molecules simultaneously to block both Wnt binding sites. Wnt isoforms in the third class (Wnt7a, 7b, and 10a) that are not antagonized by YW211.31.57 or YW210.09 antibody might bind to a site on LRP6 not blocked by either antibody or, alternatively, they may have the ability to bind to either of the Wnt-binding sites defined by the antibodies. For each of these Wnt isoforms, the combination of both antibodies also does not inhibit their signaling, but rather potentiates or does not affect their activity, suggesting that these Wnt isoforms can bind a site that is distinct from the YW211.31.57 and YW210.09 epitopes (Figure 3E). The observed activities of the YW211.31.57 and YW210.09 antibody combination on Wnt signaling induced by exogenous Wnt isoforms also extend to endogenous, autocrine Wnt signaling. In PA-1 (Figure 1D) and Ntera-2 (data not shown) cell lines, in which YW211.31.57 antibody inhibits and YW210.09 potentiates autocrine Wnt signaling, the antibody combination inhibits signaling. In Hs578T and EKVX cells, where both antibodies potentiate autocrine Wnt signaling, the antibody combination also potentiates (Figure 5B and 5C). LRP6 Antibodies Differentially Inhibit Wnt Binding to Multiple Sites The reciprocal activities of the two LRP6 antibodies suggest that YW211.31.57 and YW210.09 interact with distinct Wnt binding sites on LRP6, and that Wnt binding was competed by the antagonist antibody but allowed by the potentiating antibody. Bourhis et al. (2010) previously developed a biolayer interferometry assay that measures purified Wnt proteins binding to purified, immobilized LRP6 extracellular domain protein fragments [14]. This assay demonstrated that Wnt3a binds to the E3-E4 region of LRP6, where the epitope for YW211.31.57 antibody resides, and Wnt9b (in the same class as Wnt1 for antibody interactions) binds only to the E1-E2 region, where YW210.09 antibody also binds. Here we show that YW211.31.57 antibody, but not YW210.09, inhibits binding of Wnt3a to the LRP6 E1-E4 protein fragment (Figure 6A). Conversely, YW210.09 but not YW211.31.57 inhibits Wnt9b binding to E1-E4 (Figure 6B). Antibody-mediated inhibition of Wnt binding can also be detected using the smaller Wnt-binding fragments, E3-E4 for Wnt3a and E1-E2 for Wnt9b (Figure 6C and 6D). The one-armed YW211.31 antibody can also inhibit Wnt3a binding to the E3-E4 fragment (Figure 6C). Also, YW211.31.57 and YW210.09 can be bound sequentially to LRP6 PLoS ONE | www.plosone.org 7 September 2010 | Volume 5 | Issue 9 | e12682