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Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling

Abstract

The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues.

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Figure 1: Conditional deletion of Lgr4 and Lgr5.
Figure 2: Wnt target genes are downstream of Lgr4/5.
Figure 3: LGR4 is essential for transmitting RSPO1 signals but dispensable for transmitting WNT3A signals.
Figure 4: Direct physical interaction of RSPO1 with LGR4/5/6 exodomains.
Figure 5: Rescue of Lgr4/5 deletion in cultured crypt organoids by Wnt signals.

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Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data have been deposited in the GEO database under accession number GSE28265. Mass spectrometry data sets are available at ProteomeCommons.org Tranche Repository https://proteomecommons.org/tranche/data-downloader.jsp?h52LOW5tCJBOfT%2FpcCAtMrPqCgTTOd247s6poPgSvwu16KiVwCfExWdJ0jifGdI4FraidTHUnl1PYhIoT0nTs1zdwKmKEAAAAAAAACzw%3D%3D.

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Acknowledgements

We thank G. Vassart for Lgr4-LacZ intestinal tissue, D. Winton for Ah-Cre mice, S. Robine for Villin-CreERT2 mice, A. Moerkamp and C. Verheul for experimental help and H. Farin for figures.

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All Hubrecht Institute authors performed experiments under guidance of H.C.; S.M., A.J.R.H. and T.Y.L. performed mass spectrometry; P.K. and P.J.P. performed electron microscopy analysis; R.B.M.S. performed plasmon surface resonance; and Y.M. and K.N. generated the Lgr4 knockout mouse.

Corresponding author

Correspondence to Hans Clevers.

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The authors declare competing financial interests as inventors on several patents relating to this work.

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de Lau, W., Barker, N., Low, T. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011). https://doi.org/10.1038/nature10337

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