Fig. 4: LOF missense mutants in ZNRF3 R-Spondin domain fail to reach the membrane correctly.
From: Lack of dominant-negative activity for tumor-related ZNRF3 missense mutations at endogenous levels
Analysis of plasma membrane presence of selected R-Spondin and RING domain ZNRF3 variants. A Schematic overview showing the process of biotinylation and IP experiments. The Sulfo-NHS-LC-Biotin reagent is added to live cells for cell membrane protein labeling, followed by α-FLAG beads immunoprecipitation of ZNRF3/RNF43 FLAG-tagged protein. Next, IRDye800CW-streptavidin is used to label Biotin to measure the protein levels of ZNRF3/RNF43 reaching the cell membrane. B In contrast to wild-type ZNRF3 and non-defective R-Spondin domain variants, six tested variants defective in β-catenin regulation (red squares) fail to reach the membrane correctly. Defective RING domain variants (I295T to C333S) have no problems in reaching the membrane and are detected at increased levels in line with their increased overall stability. C HeLa cells were transfected with ZNRF3 variants. Expression and distribution of ZNRF3 variants was 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. The ZNRF3 WT and R149Q variants are capable of reaching the cell membrane, while G150V, P179L, and I205T, which lead to the loss of function of ZNRF3, cannot be detected at the cell membrane.
