Alternative titles; symbolsG PROTEIN-COUPLED RECEPTOR 49; GPR49HGNC Approved Gene Symbol: LGR5Cytogenetic location: 12q21.1 Genomic coordinates (GRCh38): 12:...
Alternative titles; symbols
HGNC Approved Gene Symbol: LGR5
Cytogenetic location: 12q21.1 Genomic coordinates (GRCh38): 12:71,439,797-71,586,309 (from NCBI)
The receptors for glycoprotein hormones such as follicle-stimulating hormone (FSH; see 136530) and thyroid-stimulating hormone (TSH; see 188540) are G protein-coupled, 7-transmembrane receptors (GPCRs) with large N-terminal extracellular domains. Leucine-rich repeat (LRR)-containing GPCRs (LGRs) form a subgroup of the GPCR superfamily (summary by McDonald et al., 1998).
▼ Cloning and Expression
By EST database searching with known GPCRs as queries, followed by modified RACE-PCR, McDonald et al. (1998) identified a cDNA encoding GPR49, which they called HG38. The deduced 907-amino acid 7-transmembrane GPR49 protein has an N-terminal signal peptide, 16 LRRs, and is approximately 35% identical to FSH receptor (136435), TSH receptor (603372), and luteinizing hormone (LH) receptor (152790). Functional analysis, however, did not show activation of GPR49 by any ligands of those receptors. Northern blot analysis revealed expression of a 5.0-kb GPR49 transcript in skeletal muscle, spinal cord, and various subregions of brain.
Hsu et al. (1998) also cloned GPR49, which they termed LGR5. Sequence analysis predicted that LGR5 contains a signal peptide; N- and C-flanking cysteine-rich sequences separated by 17 LRRs; 4 potential N-glycosylation sites; a transmembrane region; and an 82-residue cytoplasmic tail with a conserved potential phosphorylation site and potential SH2- and SH3-interacting sequences. Northern blot analysis detected major 4.3-kb and minor 2.4-kb LGR5 transcripts that were most abundant in skeletal muscle, with lower amounts in placenta, spinal cord, brain, adrenal, colon, stomach, and bone marrow. Functional analysis showed that expression of a chimeric receptor composed of the extracellular domain of LH receptor with the transmembrane and cytoplasmic domains of LGR5 resulted in binding of hCG (CGB; 118860) but no increase in basal production of cAMP, suggesting that LGR5 may signal through another mechanism.
▼ Gene Function
Barker et al. (2007) selected LGR5 from a panel of intestinal Wnt target genes for its restricted crypt expression. Using 2 knockin alleles, Barker et al. (2007) revealed exclusive expression of mouse Lgr5 in cycling columnar cells at the crypt base. In addition, Lgr5 was expressed in rare cells in several other tissues. Using an inducible Cre knockin allele and the Rosa26-lacZ reporter strain, lineage-tracing experiments were performed in adult mice. The Lgr5-positive crypt base columnar cell generated all epithelial lineages over a 60-day period, suggesting that it represents the stem cell of the small intestine and colon. Barker et al. (2007) concluded that the expression pattern of LGR5 suggests that it marks stem cells in multiple adult tissues and cancers.
In mice, Jaks et al. (2008) found that Lgr5 was expressed in actively cycling cells in the bulge and secondary germ of telogen hair follicles and in the lower outer root sheath of anagen hair follicles. Lgr5-positive cells were an actively proliferating and multipotent stem cell population that gave rise to new hair follicles and maintained all cell lineages of the hair follicle over long periods of time. Lgr5-positive progeny repopulated other stem cell compartments in the hair follicle, supporting a stem or progenitor cell hierarchy. By marking Lgr5-positive cells during trafficking through the lower outer root sheath, Jaks et al. (2008) showed that these cells retained stem cell properties and contributed to hair follicle growth during the next anagen. Expression analysis suggested involvement of autocrine Hedgehog (SHH; 600725) signaling in maintaining the Lgr5-positive stem cell population.
Using the LGR knockin mouse model developed by Barker et al. (2007), Barker et al. (2009) demonstrated that the deletion of APC (611731) in the stem cells marked by Lgr5 leads to their transformation within days. Transformed stem cells remained located at crypt bottoms while fueling a growing microadenoma. These microadenomas showed unimpeded growth and developed into macroscopic adenomas within 3 to 5 weeks. The distribution of Lgr5-positive cells within stem cell-derived adenomas indicated that a stem cell/progenitor cell hierarchy is maintained in early neoplastic lesions. When Apc was deleted in short-lived transit-amplifying cells using a different Cre mouse, the growth of the induced microadenomas rapidly stalled. Even after 30 weeks, large adenomas were very rare in these mice. Barker et al. (2009) concluded that stem cell-specific loss of APC results in progressively growing neoplasia.
Sato et al. (2009) described the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultaneously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5+ stem cells can also initiate these crypt-villus organoids. Tracing experiments indicated that the Lgr5+ stem cell hierarchy is maintained in organoids. Sato et al. (2009) concluded that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a nonepithelial cellular niche.
Sato et al. (2011) found a close physical association of Lgr5 stem cells with Paneth cells in mice, both in vivo and in vitro. Cd24 (600074)+ Paneth cells express Egf (131530), Tgf-alpha (190170), Wnt3 (165330), and the Notch ligand Dll4 (605185), all essential signals for stem cell maintenance in culture. Coculturing of sorted stem cells with Paneth cells markedly improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. Sato et al. (2011) concluded that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.
De Lau et al. (2011) found that conditional deletion of LGR5 and LGR4 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 demonstrated that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the 4 R-spondins (RSPO1, 609595; RSPO2, 610575; RSPO3, 610574; and RSPO4, 610573), secreted Wnt pathway agonists, can bind to Lgr4, Lgr5, and Lgr6 (606653). In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A (606359). Removal of LGF4 does not affect WNT3A signaling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by reexpression of LGR4, LGR5, or LGR6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologs are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins.
Using a human diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus, Tian et al. (2011) specifically ablated Lgr5-expressing cells in mice and found that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium, indicating that other cell types can compensate for the elimination of this population. After ablation of Lgr5-expressing cells, progeny production by Bmi1 (164831)-expressing cells increased, indicating that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Indeed, lineage tracing showed that Bmi1-expressing cells gave rise to Lgr5-expressing cells, pointing to a hierarchy of stem cells in the intestinal epithelium. Tian et al. (2011) concluded that their results demonstrated that Lgr5-expressing cells are dispensable for normal intestinal homeostasis, and that in the absence of these cells, Bmi1-expressing cells can serve as an alternative stem cell pool. Tian et al. (2011) suggested that the Bmi1-expressing stem cells may represent both a reserve stem cell pool in case of injury to the small intestine epithelium and a source for replenishment of the Lgr5-expressing cells under nonpathologic conditions.
Studying mouse models, Schepers et al. (2012) provided direct, functional evidence for the presence of stem cell activity within primary intestinal adenomas, a precursor to intestinal cancer. By lineage retracing using the multicolor Cre reporter R26R-Confetti, they demonstrated that the crypt stem cell marker Lgr5 also marks a subpopulation of adenoma cells that fuel the growth of established intestinal adenomas. These Lgr5+ cells, which represent about 5 to 10% of the cells in the adenomas, generate additional Lgr5+ cells as well as all other adenoma cell types. The Lgr5+ cells are intermingled with Paneth cells near the adenoma base, a pattern reminiscent of the architecture of the normal crypt niche.
Huch et al. (2013) showed that Lgr5-lacZ is not expressed in healthy adult liver; however, small Lgr5-LacZ-positive cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signaling. As shown by mouse lineage tracing using a Lgr5-IRES-creERT2 knockin allele, damage-induced Lgr5+ cells generate hepatocytes and bile ducts in vivo. Single Lgr5+ cells from damaged mouse liver can be clonally expanded as organoids in Rspo1 (609595)-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah (613871)-null mice. Huch et al. (2013) concluded that previous observations concerning Lgr5+ stem cells in actively self-renewing tissues can be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.
Shimokawa et al. (2017) showed that human LGR5-positive colorectal cancer cells serve as cancer stem cells (CSCs) in growing cancer tissues. Lineage-tracing experiments with a tamoxifen-inducible Cre knockin allele of LGR5 revealed the self-renewal and differentiation capacity of LGR5+ tumor cells. Selective ablation of LGR5+ CSCs in LGR5-iCaspase9 knockin organoids led to tumor regression, followed by tumor regrowth driven by reemerging LGR5+ cancer stem cells. A KRT20 knockin reporter marked differentiated cancer cells that constantly diminish in tumor tissues, while reverting to LGR5+ cancer stem cells and contributing to tumor regrowth after LGR5+ CSC ablation. Shimokawa et al. (2017) also showed that combined chemotherapy potentiates targeting of LGR5+ cancer stem cells.
Using unbiased quantitative lineage-tracing approaches, biophysical modeling, and intestinal transplantation to investigate the role of fetal LGR5-positive cells in the establishment of the adult intestinal stem cell population, Guiu et al. (2019) demonstrated that all cells of the mouse intestinal epithelium, irrespective of their location and pattern of LGR5 expression in the fetal gut tube, contribute actively to the adult intestinal stem cell pool. Using 3D imaging, Guiu et al. (2019) found that during fetal development the villus undergoes gross remodeling and fission. This brings epithelial cells from the nonproliferative villus into the proliferative intervillus region, which enables them to contribute to the adult stem cell niche. The results demonstrated that large-scale remodeling of the intestinal wall and cell-fate specification are closely linked. Moreover, these findings provided a direct link between the observed plasticity and cellular reprogramming of differentiating cells in adult tissues following damage, revealing that stem cell identity is an induced rather than a hardwired property.
By radiation hybrid analysis, McDonald et al. (1998) mapped the GPR49 gene to chromosome 12q22-q23, a region with several genes involved in muscle growth and development. By FISH, Hsu et al. (1998) mapped the GPR49 gene to chromosome 12q15.
▼ Animal Model
Quigley et al. (2009) integrated germline polymorphisms with gene expression in normal skin from a M. musculus x M. spretus backcross to generate a network view of the gene expression architecture of mouse skin. They identified expression motifs that contribute to tissue organization and biologic functions related to inflammation, hematopoiesis, cell cycle control, and tumor susceptibility. Motifs associated with inflammation, epidermal barrier function, and proliferation were differentially regulated in backcross mice susceptible or resistant to tumor development. The intestinal stem cell marker Lgr5 was identified as a candidate master regulator of the hair follicle.