Fig. 3: Analysis of missense variants within the ZNRF3 RING domain.
From: Lack of dominant-negative activity for tumor-related ZNRF3 missense mutations at endogenous levels
About half of the tested RING domain variants result in a (partial) loss-of-function or hyperactivating behavior of ZNRF3. A A β-catenin reporter assay reveals 14/27 variants that show a significant loss of β-catenin regulatory activity. Wnt3A conditioned medium was added to Empty Vector (EV) and all ZNRF3 variants 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 (****P < 0.0001). B Diagram depicting the RING domain structure of ZNRF3. The green circles are the residues required for interaction with ubiquitin-conjugating E2 enzymes, while yellow circles are the residues involved in Zinc (Zn) coordination. Tumor-associated variants that disrupt the β-catenin regulatory function of ZNRF3 are encircled by a red border. C All variants were co-transfected with EGFP plasmids. Immunoblot shows that all defective variants show at least a 1.5-fold increased protein stability, with the exception of E305K and D321N. All relative protein stabilities are depicted as ZNRF3 variants/GFP ratios, in which the value obtained from ZNRF3-WT was set to 1.
