Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) is produced in a broad spectrum of mouse embryonic and adult tissues and its deficiency results in embryonal or perinatal lethality. The LGR4 function was mainly related to its potentiation of canonical Wnt signaling; however, several recent studies associate LGR4 with additional signaling pathways. To obtain a suitable tool for studying the signaling properties of Lgr4, we generated a tagged variant of the Lgr4 receptor using gene targeting in the mouse oocyte. The modified Lgr4 allele expresses the Lgr4 protein fused with a triple hemagglutinin (3HA) tag located at the extracellular part of the protein. The allele is fully functional, enabling tracking of Lgr4 expression in the mouse tissues. We also show that via surface labeling, the 3HA tag allows direct isolation and analysis of living Lgr4-positive cells obtained from the small intestinal crypts. Finally, the HA tag-specific antibody can be employed to characterize the biochemical features of Lgr4 and to identify possible biding partners of the protein in cells derived from various mouse tissues.

Institute of Molecular Genetics Academy of Sciences of the Czech Republic Videnska 1083 142 20 Prague 4 Czechia

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Barker N, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007;449:1003–1007. doi: 10.1038/nature06196. PubMed DOI

Barker N, et al. Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell. 2010;6:25–36. doi: 10.1016/j.stem.2009.11.013. PubMed DOI

Barker N, et al. Lgr5(+ve) stem/progenitor cells contribute to nephron formation during kidney development. Cell Rep. 2012;2:540–552. doi: 10.1016/j.celrep.2012.08.018. PubMed DOI

Buczacki SJ, Zecchini HI, Nicholson AM, Russell R, Vermeulen L, Kemp R, Winton DJ. Intestinal label-retaining cells are secretory precursors expressing Lgr5. Nature. 2013;495:65–69. doi: 10.1038/nature11965. PubMed DOI

Carmon KS, Gong X, Lin Q, Thomas A, Liu Q. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. Proc Natl Acad Sci U S A. 2011;108:11452–11457. doi: 10.1073/pnas.1106083108. PubMed DOI PMC

Carmon KS, Gong X, Yi J, Thomas A, Liu Q. RSPO–LGR4 functions via IQGAP1 to potentiate Wnt signaling. Proc Natl Acad Sci U S A. 2014;111:E1221–E1229. doi: 10.1073/pnas.1323106111. PubMed DOI PMC

Cermak T, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011;39:e82. doi: 10.1093/nar/gkr218. PubMed DOI PMC

Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics MCP. 2014;13:2513–2526. doi: 10.1074/mcp.M113.031591. PubMed DOI PMC

de Lau W, et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature. 2011;476:293–297. doi: 10.1038/nature10337. PubMed DOI

de Visser KE, et al. Developmental stage-specific contribution of LGR5(+) cells to basal and luminal epithelial lineages in the postnatal mammary gland. J Pathol. 2012;228:300–309. doi: 10.1002/path.4096. PubMed DOI

Deng C, Reddy P, Cheng Y, Luo CW, Hsiao CL, Hsueh AJ. Multi-functional norrin is a ligand for the LGR4 receptor. J Cell Sci. 2013;126:2060–2068. doi: 10.1242/jcs.123471. PubMed DOI PMC

Doyle EL, Booher NJ, Standage DS, Voytas DF, Brendel VP, Vandyk JK, Bogdanove AJ. TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res. 2012;40:W117–W122. doi: 10.1093/nar/gks608. PubMed DOI PMC

Flemr M, Malik R, Franke V, Nejepinska J, Sedlacek R, Vlahovicek K, Svoboda P. A retrotransposon-driven dicer isoform directs endogenous small interfering RNA production in mouse oocytes. Cell. 2013;155:807–816. doi: 10.1016/j.cell.2013.10.001. PubMed DOI

Glinka A, et al. LGR4 and LGR5 are R-spondin receptors mediating Wnt/beta-catenin and Wnt/PCP signalling. EMBO Rep. 2011;12:1055–1061. doi: 10.1038/embor.2011.175. PubMed DOI PMC

Hao HX, et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature. 2012;485:195–200. doi: 10.1038/nature11019. PubMed DOI

Hebert AS, Richards AL, Bailey DJ, Ulbrich A, Coughlin EE, Westphall MS, Coon JJ. The one hour yeast proteome. Mol Cell Proteomics MCP. 2014;13:339–347. doi: 10.1074/mcp.M113.034769. PubMed DOI PMC

Hsu SY, Liang SG, Hsueh AJ. Characterization of two LGR genes homologous to gonadotropin and thyrotropin receptors with extracellular leucine-rich repeats and a G protein-coupled, seven-transmembrane region. Mol Endocrinol. 1998;12:1830–1845. doi: 10.1210/mend.12.12.0211. PubMed DOI

Hsu PJ, Wu FJ, Kudo M, Hsiao CL, Hsueh AJ, Luo CW. A naturally occurring Lgr4 splice variant encodes a soluble antagonist useful for demonstrating the gonadal roles of Lgr4 in mammals. PLoS ONE. 2014;9:e106804. doi: 10.1371/journal.pone.0106804. PubMed DOI PMC

Jaks V, Barker N, Kasper M, van Es JH, Snippert HJ, Clevers H, Toftgard R. Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet. 2008;40:1291–1299. doi: 10.1038/ng.239. PubMed DOI

Kasparek P, Krausova M, Haneckova R, Kriz V, Zbodakova O, Korinek V, Sedlacek R. Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases. FEBS Lett. 2014;588:3982–3988. doi: 10.1016/j.febslet.2014.09.014. PubMed DOI

Kato S, Mohri Y, Matsuo T, Ogawa E, Umezawa A, Okuyama R, Nishimori K. Eye-open at birth phenotype with reduced keratinocyte motility in LGR4 null mice. FEBS Lett. 2007;581:4685–4690. doi: 10.1016/j.febslet.2007.08.064. PubMed DOI

Kinzel B, et al. Functional roles of Lgr4 and Lgr5 in embryonic gut, kidney and skin development in mice. Dev Biol. 2014;390:181–190. doi: 10.1016/j.ydbio.2014.03.009. PubMed DOI

Krausova M, Korinek V. Wnt signaling in adult intestinal stem cells and cancer. Cell Signal. 2014;26:570–579. doi: 10.1016/j.cellsig.2013.11.032. PubMed DOI

Leighton PA, et al. Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature. 2001;410:174–179. doi: 10.1038/35065539. PubMed DOI

Li C, et al. Simultaneous gene editing by injection of mRNAs encoding transcription activator-like effector nucleases into mouse zygotes. Mol Cell Biol. 2014;34:1649–1658. doi: 10.1128/MCB.00023-14. PubMed DOI PMC

Liu S et al. (2013) Lgr4 gene deficiency increases susceptibility and severity of dextran sodium sulfate-induced inflammatory bowel disease in mice. J Biol Chem 288:8794–8803; discussion 8804. doi:10.1074/jbc.M112.436204 PubMed PMC

Luo J, et al. LGR4 is a receptor for RANKL and negatively regulates osteoclast differentiation and bone resorption. Nat Med. 2016;22:539–546. doi: 10.1038/nm.4076. PubMed DOI

Masuda T, Tomita M, Ishihama Y. Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res. 2008;7:731–740. doi: 10.1021/pr700658q. PubMed DOI

Mazerbourg S, et al. Leucine-rich repeat-containing, G protein-coupled receptor 4 null mice exhibit intrauterine growth retardation associated with embryonic and perinatal lethality. Mol Endocrinol. 2004;18:2241–2254. doi: 10.1210/me.2004-0133. PubMed DOI

McDonald T, Wang R, Bailey W, Xie G, Chen F, Caskey CT, Liu Q. Identification and cloning of an orphan G protein-coupled receptor of the glycoprotein hormone receptor subfamily. Biochem Biophys Res Commun. 1998;247:266–270. doi: 10.1006/bbrc.1998.8774. PubMed DOI

Mendive F, Laurent P, Van Schoore G, Skarnes W, Pochet R, Vassart G. Defective postnatal development of the male reproductive tract in LGR4 knockout mice. Dev Biol. 2006;290:421–434. doi: 10.1016/j.ydbio.2005.11.043. PubMed DOI

Mohri Y, Kato S, Umezawa A, Okuyama R, Nishimori K. Impaired hair placode formation with reduced expression of hair follicle-related genes in mice lacking Lgr4. Dev Dyn. 2008;237:2235–2242. doi: 10.1002/dvdy.21639. PubMed DOI

Mustata RC, Van Loy T, Lefort A, Libert F, Strollo S, Vassart G, Garcia MI. Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells ex vivo. EMBO Rep. 2011;12:558–564. doi: 10.1038/embor.2011.52. PubMed DOI PMC

Plaks V, et al. Lgr5-expressing cells are sufficient and necessary for postnatal mammary gland organogenesis. Cell Rep. 2013;3:70–78. doi: 10.1016/j.celrep.2012.12.017. PubMed DOI PMC

Planas-Paz L, et al. The RSPO–LGR4/5–ZNRF3/RNF43 module controls liver zonation and size. Nat Cell Biol. 2016;18:467–479. doi: 10.1038/ncb3337. PubMed DOI

Ruffner H, et al. R-spondin potentiates Wnt/beta-catenin signaling through orphan receptors LGR4 and LGR5. PLoS ONE. 2012;7:e40976. doi: 10.1371/journal.pone.0040976. PubMed DOI PMC

Sato T, Clevers H. Primary mouse small intestinal epithelial cell cultures. Methods Mol Biol. 2013;945:319–328. doi: 10.1007/978-1-62703-125-7_19. PubMed DOI

Snippert HJ, et al. Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science. 2010;327:1385–1389. doi: 10.1126/science.1184733. PubMed DOI

Tyanova S, et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods. 2016;13:731–740. doi: 10.1038/nmeth.3901. PubMed DOI

Valenta T, Lukas J, Doubravska L, Fafilek B, Korinek V. HIC1 attenuates Wnt signaling by recruitment of TCF-4 and beta-catenin to the nuclear bodies. EMBO J. 2006;25:2326–2337. doi: 10.1038/sj.emboj.7601147. PubMed DOI PMC

van de Wetering M, et al. The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell. 2002;111:241–250. doi: 10.1016/S0092-8674(02)01014-0. PubMed DOI

VanDussen KL, Samuelson LC. Mouse atonal homolog 1 directs intestinal progenitors to secretory cell rather than absorptive cell fate. Dev Biol. 2010;346:215–223. doi: 10.1016/j.ydbio.2010.07.026. PubMed DOI PMC

Yan KS, et al. Non-equivalence of Wnt and R-spondin ligands during Lgr5 + intestinal stem-cell self-renewal. Nature. 2017;545:238–242. doi: 10.1038/nature22313. PubMed DOI PMC

Yee KK, Li Y, Redding KM, Iwatsuki K, Margolskee RF, Jiang P. Lgr5–EGFP marks taste bud stem/progenitor cells in posterior tongue. Stem Cells. 2013;31:992–1000. doi: 10.1002/stem.1338. PubMed DOI PMC

Yi J, Xiong W, Gong X, Bellister S, Ellis LM, Liu Q. Analysis of LGR4 receptor distribution in human and mouse tissues. PLoS ONE. 2013;8:e78144. doi: 10.1371/journal.pone.0078144. PubMed DOI PMC

Msx1 loss suppresses formation of the ectopic crypts developed in the Apc-deficient small intestinal epithelium