ZNRF3 and RNF43 are active monomeric E3 ubiquitin ligases that self-associate

The WNT signaling pathway regulates cell proliferation and stem cell maintenance. Its sustained and inappropriate activation results in excessive cell division and cancer. WNT signaling is activated upon interaction of the soluble WNT ligand with the Frizzled (FZD) receptor. The RING E3 ubiquitin ligases RNF43 and ZNRF3 promote the ubiquitylation and internalization of FZD, thereby turning off WNT signaling, and their inactivation causes cancer. Here, we identified the determinants of ubiquitin transfer by ZNRF3 and RNF43 and report the structure of the RING domain from ZNRF3. We found that the RING domain was monomeric and that RING dimerization was not required for its ubiquitin ligase activity. However, the ectodomain of ZNRF3 dimerizes, and our data supported a model in which the cytoplasmic domains are in close proximity in cells and interact, even though RING dimerization was not required for ubiquitin transfer. Our studies provide a framework for understanding how the E3 ubiquitin ligase activity of ZNRF3 and RNF43 is regulated.