Supplementary MaterialsSupplementary Figures. the ASGR1-RSPO2 proteins activated Wnt signaling (Fig.?1C right panel). In the presence of Wnt3a conditioned media in HEK293 cells, ASGR1-RSPO2-WT enhanced Wnt3a activity in a dose-dependent manner, like GFP-RSPO2-WT, while both ASGR1-RSPO2-RA and ASGR1-RSPO2-5mut were inactive (Fig.?1C). In contrast, in HuH-7 cells which expresses (verified by quantitative PCR analysis, Fig.?1D), ASGR1-RSPO2-RA significantly enhanced Wnt3a-induced signaling (Fig.?1C lower left panel) while ASGR1-RSPO2-5mut did not. Therefore, enhanced Wnt3a-induced signaling elicited by ASGR1-RSPO2-RA depends on its ability to bind E3 ligases. ASGR1-RSPO2-WT also showed a?~?sixfold increase in Wnt3a-induced signaling compared to GFP-RSPO2-WT (Fig.?1C), suggesting that this attachment MW-150 hydrochloride of the ASGR1 scFv may have synergized with LGR to further enhance the WT RSPO2 function. In addition to the STF reporter assay, Western blots were used to examine Wnt signaling pathway components directly. As shown in Figs.?1E and S2, as the treatment of HuH-7 cells with Wnt3a conditioned media did not induce a significant switch in phosphorylation of important proteins of the Wnt signaling pathway, treatment with GFP-RSPO2-WT enhanced the Wnt3a-signalling and significantly increased levels of both phosphorylated LRP6 and DVL2 proteins, in addition to increasing total LRP6 protein levels. The mutations in Fu2 abolished any enhanced Wnt3a-signaling that was seen in the GFP-RSPO2-RA treated cells. However, the fusion to ASGR scFv (ASGR1-RSPO2-RA) rescued the loss of function phenotype seen with GFP-RSPO2-RA, slightly increased total LRP6 protein and phosphorylated DVL2 levels, and significantly increased levels of phosphorylated LRP6 (Figs.?1E and S2), suggesting that ASGR1-RSPO2-RA and RSPO2-WT amplified Wnt signaling to a similar extent. Validation of specificity by over expression of MW-150 hydrochloride the targeted receptor To further confirm that the cell-specific activity of -ASGR1-RSPO2-RA was dependent on the presence of the targeted ASGR1, we transiently transfected HEK293 cells with a plasmid encoding a full length human cDNA. Since ASGR1 and ASGR2 form a complex and neither are MW-150 hydrochloride expressed in HEK293 cells (Fig.?1D), HEK293 cells were also co-transfected with both cDNAs. In addition, an unrelated receptor TFR1 (encoded by a cDNA) was transfected as a negative control. Like the un-transfected parental HEK293 cells, only the RSPO2-WT mimetics made up of either GFP or ASGR1 enhanced Wnt signaling in a Wnt-dependent manner in cells transfected with (compare Fig.?1C top panels to Fig.?2A top panels). In contrast, in cells transfected with either alone or co-transfected with both and expression alone was sufficient to support the activity of ASGR1-RSPO2-RA, because the ASGR1 subunit can go through endocytosis independently of ASGR222. The fusion of ASGR1 also increased the potency of RSPO2-WT protein, similar to that observed in HuH-7 cells (compare Fig.?1C lower panels to Fig.?2A lower panels). Activity of ASGR1-RSPO mimetics was entirely dependent on the presence of Wnt ligand (Fig.?2A right panels). The ASGR1-dependence of the ASGR1-RSPO mimetics was also confirmed in another ASGR1 unfavorable cell collection, A431, which is derived from a human epidermoid carcinoma and expresses genes at very low levels (Fig.?1D). Comparable to what was observed in the HEK293 transfection studies, ASGR1-RSPO2-RA was only active in A431 cells expressing but MW-150 hydrochloride not in A431 cells expressing the unfavorable control receptor (Fig.?2B). Furthermore, the ASGR1 SLCO5A1 scFv also increased the potency of the MW-150 hydrochloride RSPO2-WT?fusion protein in an ASGR1-depedent manner, and the activity of all RSPO mimetics were completely dependent on the current presence of Wnt ligand (Fig.?2B correct sections). Collectively, these outcomes confirmed that RSPO-mediated downregulation of E3 ligases through binding of LGR protein can be changed by targeting.