Fig. 5: ZNRF3 R-Spondin binding domain deletions retain the ability to correctly traffic to the membrane and regulate Wnt-induced β-catenin signaling.

Comparing R-Spondin/PA domain deletions with defective missense mutations. A A β-catenin reporter assay reveals that LOF missense R-spondin domain variants are defective in reducing Wnt-induced β-catenin signaling. However, ZNRF3 with deletions in the same R-spondin binding domain still behave similar to WT. Wnt3A conditioned medium was added to Empty Vector (EV) and all ZNRF3 variant transfected cells. L-control conditioned medium was added to EV transfected cells as a negative control for β-catenin signaling. All relative β-catenin reporter activities are depicted as WRE/CMV-Renilla ratios, in which the value obtained for ZNRF3-WT was arbitrarily set to 1. Statistical significance was assessed using a t-test (*P < 0.1, **P < 0.01). B ZNRF3 variants were co-transfected with an EGFP plasmid serving as transfection control. Immunoblot shows that Δ57-207 and Δ103-205 exhibit comparable expression to WT, and Δ101-142 shows slightly reduced stability. The missense variants show strong protein instability. All relative protein stabilities are depicted as ZNRF3 variants/GFP ratios, in which the value obtained from ZNRF3-WT was set to 1. C HeLa cells were transfected with ZNRF3 variants. Expression and distribution of ZNRF3 variants were assessed by immunofluorescence. The signal of MYC (red) indicates the ZNRF3 expression on the cell membrane, and the signal of HA (green) indicates the ZNRF3 expression on cell membrane or in cytoplasm. ZNRF3 WT and all R-Spondin deletions are capable of efficiently reaching the cell membrane.