Structures of Wnt-antagonist ZNRF3 and its complex with R-spondin 1 and implications for signaling

PLoS One. 2013 Dec 12;8(12):e83110. doi: 10.1371/journal.pone.0083110. eCollection 2013.

Abstract

Zinc RING finger 3 (ZNRF3) and its homolog RING finger 43 (RNF43) antagonize Wnt signaling in adult stem cells by ubiquitinating Frizzled receptors (FZD), which leads to endocytosis of the Wnt receptor. Conversely, binding of ZNRF3/RNF43 to LGR4-6 - R-spondin blocks Frizzled ubiquitination and enhances Wnt signaling. Here, we present crystal structures of the ZNRF3 ectodomain and its complex with R-spondin 1 (RSPO1). ZNRF3 binds RSPO1 and LGR5-RSPO1 with micromolar affinity via RSPO1 furin-like 1 (Fu1) domain. Anonychia-related mutations in RSPO4 support the importance of the observed interface. The ZNRF3-RSPO1 structure resembles that of LGR5-RSPO1-RNF43, though Fu2 of RSPO1 is variably oriented. The ZNRF3-binding site overlaps with trans-interactions observed in 2:2 LGR5-RSPO1 complexes, thus binding of ZNRF3/RNF43 would disrupt such an arrangement. Sequence conservation suggests a single ligand-binding site on ZNRF3, consistent with the proposed competing binding role of ZNRF3/RNF43 in Wnt signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult Stem Cells / metabolism
  • Animals
  • Crystallography, X-Ray
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Mice
  • Multiprotein Complexes / chemistry*
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism
  • Oncogene Proteins / chemistry
  • Oncogene Proteins / genetics
  • Oncogene Proteins / metabolism
  • Protein Structure, Quaternary
  • Receptors, G-Protein-Coupled / chemistry
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism
  • Thrombospondins / chemistry*
  • Thrombospondins / genetics
  • Thrombospondins / metabolism
  • Ubiquitin-Protein Ligases / chemistry*
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism
  • Wnt Signaling Pathway*

Substances

  • DNA-Binding Proteins
  • LGR5 protein, human
  • Lgr5 protein, mouse
  • Multiprotein Complexes
  • Oncogene Proteins
  • RSPO1 protein, human
  • RSPO1 protein, mouse
  • Receptors, G-Protein-Coupled
  • Thrombospondins
  • RNF43 protein, human
  • RNF43 protein, mouse
  • Ubiquitin-Protein Ligases
  • ZNRF3 protein, human
  • Znrf3 protein, mouse

Grant support

Financial support was provided by the Netherlands Organization for Scientific Research (NWO-CW grant no. 700.57.010), the European Research Council (grant no. 233229), and the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant no. 283570). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.