Abstract
LGR4/5/6 and RNF43 interact with R-spondin cysteine-rich domains via distinct binding surfaces.
Major finding: LGR4/5/6 and RNF43 interact with R-spondin cysteine-rich domains via distinct binding surfaces.
Approach: Crystal structures provide insight into the structural mechanisms regulating WNT signaling.
Impact: Disease-associated mutations disrupt LGR–RSPO–RNF43 complex formation and impair WNT signaling.
The R-spondin (RSPO) family of secreted glycoproteins regulates stem cell function by enhancing activation of WNT/β-catenin signaling. RSPOs have recently been shown to bind the extracellular domains (ECD) of leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4), LGR5, and LGR6, as well as the transmembrane E3 ubiquitin ligases ring finger protein 43 (RNF43) and zinc and ring finger 3 (ZNRF3) to stabilize canonical WNT pathway receptors. To define the structural basis of these interactions, Wang and colleagues characterized the crystal structure of the LGR4 ECD in complex with the 2 RSPO1 furin-like cysteine-rich domains (CRD), which are required to promote WNT signaling. LGR4 used an N-terminal concave surface to interact with both RSPO1 CRDs via salt bridge and hydrophobic interactions. Mutation of critical CRD residues disrupted RSPO1 binding to LGR4 and impaired WNT activation. In a complementary study, Chen and colleagues determined the structure of the ternary complex consisting of the RSPO1 CRD fold, LGR5 ECD, and the protease-associated (PA) domain of RNF43. RSPO1 interacted with these receptors via distinct CRD binding surfaces, specifically a rod module that formed hydrophobic contacts with LGR5 and a β-hairpin protrusion that contacted the RNF43 PA domain. Furthermore, LGR5 did not directly contact RNF43, but stabilized the weak interaction between RSPO1 and RNF43, supporting its role as an engagement receptor. Mutations in key residues at the RSPO–RNF43/ZNRF3 interface were associated with developmental defects and tumorigenesis, emphasizing the role of RNF43/ZNRF3 as effector receptors. Both groups also found that the C-terminal hinge region of the LGR4/5 ECD did not interact with RSPO1 ligand, possibly explaining why, in contrast to LGR hormone receptors, LGR4/5/6 do not signal via G proteins. Together, these findings provide mechanistic insights into the role of RSPOs in regulating WNT signaling complex formation.
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