Unlike LGR4, LGR5 potentiates Wnt-β-catenin signaling without sequestering E3 ligases

Area of Science:

• Molecular and Cellular Biology
• Stem Cell Biology
• Signal Transduction

Background:

• LGR4 and LGR5 are homologous receptors crucial for organ development and stem cell maintenance.
• Both receptors bind R-spondins (RSPOs) and potentiate Wnt-β-catenin signaling, but their precise mechanisms differ.
• LGR4 is essential for intestinal stem cell self-renewal, while LGR5's role is less understood in this context.

Purpose of the Study:

• To elucidate the distinct molecular mechanisms by which LGR4 and LGR5 regulate Wnt signaling.
• To investigate the interaction of LGR4 and LGR5 with E3 ligases RNF43 and ZNRF3 in the presence or absence of RSPO.
• To determine the structural basis for differential E3 ligase interaction and signaling potentiation by LGR4 and LGR5.

Main Methods:

• Coimmunoprecipitation assays to assess protein-protein interactions.
• Proximity ligation assays and time-resolved FRET to study molecular interactions in whole cells.
• Competition binding assays and domain-swapping experiments to map interaction domains.

Main Results:

• Full-length LGR4 formed a stable complex with E3 ligases ZNRF3 and RNF43 independently of RSPO.
• LGR5 did not directly interact with ZNRF3 or RNF43, even in the presence of RSPO.
• The seven-transmembrane domain of LGR4 was identified as the key region mediating E3 ligase interaction; LGR5 interacted with Wnt signalosome components (FZD, LRP6) to enhance LRP6 phosphorylation.

Conclusions:

• LGR4's direct interaction with E3 ligases provides a mechanism for its essential role in intestinal stem cell self-renewal.
• LGR5's distinct mechanism, involving interaction with the Wnt signalosome, may explain its differential contribution to organ development and stem cell fitness.
• These findings offer a molecular basis for the distinct physiological roles of LGR4 and LGR5.