R-spondins can potentiate WNT signaling without LGR receptors

The WNT signaling pathway regulates patterning and morphogenesis during embryonic development and promotes tissue renewal and regeneration in adults. Some WNT responses in vertebrates depend on a second signal provided by the R-spondin family of four secreted proteins (RSPO1-4) that drive the renewal of stem cells in many tissues. RSPOs markedly amplify target cell sensitivity to WNT ligands by neutralizing two transmembrane E3 ligases, ZNRF3 and RNF43, which reduce cell-surface levels of WNT receptors. Chromosomal translocations that increase RSPO expression or that inactivate ZNRF3/RNF43 can drive human cancers. RSPOs contain tandem furin-like repeats (FU1 and FU2), a thrombospondin type I (TSP) domain, and a basic region (BR). RSPOs simultaneously engage ZNRF3/RNF43 through their FU1 domain and one of three leucine-rich repeat-containing G-protein coupled receptors (LGR4-6) through their FU2 domain, triggering the clearance of ZNRF3/RNF43 and the consequent rise in WNT receptor levels. LGRs are selectively expressed in various tissue stem cells and are considered the primary high-affinity receptors for RSPOs. Using purified mutant and chimeric RSPOs and cell lines lacking various receptors, we show that RSPO2 and RSPO3, but not RSPO1 and RSPO4, can potentiate WNT/β-catenin signaling in the absence of all three LGRs. The ZNRF3/RNF43-interacting FU1 domain was necessary for LGR-independent signaling, while the LGR-interacting FU2 domain was dispensable. The FU1 domain of RSPO3 was also sufficient to confer LGR-independence when transplanted to RSPO1, demonstrating that its interaction with ZNRF3/RNF43 dictates LGR-independent signaling. The enigmatic TSP/BR domains of RSPOs and their interaction with heparan sulfate proteoglycans (HSPGs), previously considered dispensable for WNT/β-catenin signaling, became essential in the absence of LGRs. These results define two alternative modes of RSPO-mediated signaling that share a common dependence on ZNRF3/RNF43, but differ in their use of either LGRs or HSPGs, with implications for understanding their mechanism of action, biological functions and evolutionary origins.