LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling

Abstract

R-spondins are secreted Wnt signalling agonists, which regulate embryonic patterning and stem cell proliferation, but whose mechanism of action is poorly understood. Here we show that R-spondins bind to the orphan G-protein-coupled receptors LGR4 and LGR5 by their Furin domains. Gain- and loss-of-function experiments in mammalian cells and Xenopus embryos indicate that LGR4 and LGR5 promote R-spondin-mediated Wnt/β-catenin and Wnt/PCP signalling. R-spondin-triggered β-catenin signalling requires Clathrin, while Wnt3a-mediated β-catenin signalling requires Caveolin-mediated endocytosis, suggesting that internalization has a mechanistic role in R-spondin signalling.

Figure 1

LGR4 and LGR5 are new regulators of Wnt and R-spondin signalling. (A–F) Wnt luciferase reporter assays in HEK293T cells stimulated with the indicated constructs or Wnt3a and/or Rspo3-ΔC, or Rspo3-ΔC+ΔTSP, or Rspo3-ΔC+ΔFurin-conditioned medium, in the presence of the indicated small interfering RNAs. Co, control medium. RLA, relative luciferase activity. TSP, thrombospondin. Error bars indicate s.d. values, n=3; ^* indicates P<0.001 by Student's t-test.

Figure 2

R-spondins bind to LGR4 and LGR5. (A) Cell surface binding assay. Cells were transfected with the indicated plasmids and subjected to binding assays with conditioned medium containing alkaline-phosphatase (AP) fusion proteins of R-spondins-ΔC, Dkk1 or hRspo3-ΔC deletion constructs (ΔTSP and ΔFurin). (B,C) Scatchard plot analysis of in vitro binding assay using LGR4, LGR5 and hRspo3-ΔC–AP. Co, control medium; SDC4, syndecan 4; TSP, thrombospondin.

Figure 3

Clathrin endocytosis is required for Rspo3 signalling. (A–L) Clathrin is colocalized with Rspo3 and is required for Rspo3 internalization. Confocal microscopy of HepG2 cells treated for 1 h with horseradish peroxidase (HRP)-tagged Rspo3-ΔC on ice (A), or at 37°C (B,D–L), or in the presence of Flag-tagged Rspo3-ΔC (C). HRP-tagged Rspo3-ΔC was visualized by tyramide signal amplification without or with coimmunofluorescence of anti-green fluorescent protein (GFP) antibody against overexpressed xtLGR4-EYFP (D), Clathrin–GFP (E) and Caveolin–GFP (F). Where indicated, cells were pretreated with small interfering RNAs (siRNAs) for 4 days (B,G–I) or with endocytic inhibitors for 1 h (J–L). (M) Clathrin is required for Rspo3-induced nuclear β-catenin accumulation. Confocal microscopy of NTERA2 cells incubated for 1 h with endocytic inhibitors or pretreated with the indicated siRNAs for 3 days. Cells were then treated for 4 h with Wnt3a, or Wnt3a together with Rspo3, or 50 mM LiCl. Scale bars indicate 10 μm. MDC, monodansylcadaverine.

Figure 4

LGR4 and LGR5 are required for Wnt/PCP signalling in Xenopus. (A,B) Rspo3 signalling requires LGR4 to induce gastrulation defects. Embryos were injected equatorially at 4-cell stage with morpholinos and/or messenger RNA (mRNA) as indicated (250 pg xRspo3 mRNA, 10 ng (+) and 20 ng (++) of LGR4 Mo1). Scale bar indicates 500 μm. (C–E) ATF2-luciferase reporter activity is reduced by LGR4/5 Mos in Xenopus embryos. Embryos were injected equatorially with ATF2-luc reporter (100 pg) and Renilla reporter plasmids (25 pg) and the indicated morpholinos (20 ng LGR4 Mo1, 5 ng LGR5 Mo1 in E) and mRNAs. Luciferase reporter assays were performed from whole embryos collected at gastrula stage. Luciferase activity in embryos injected with either Co Mo or Co Mo plus mRNA of the indicated activators within each condition were set to 100%. RLA, relative luciferase activity. All experiments were performed at least twice with three replicates each. Co, control medium; Mo, morpholino oligonucleotide.