LGR4 (Leucine-Rich Repeat G-Protein Coupled Receptor 4)
Leucine-rich repeat G-protein-coupled receptors (LGRs) are a class of G-protein-coupled receptors characterized by a large extracellular domain containing several leucine-rich repeats (LRR) (Van Loy et al. 2008). According to phylogenetic analysis, there are three known LGR subgroups in mammals (Van Loy et al. 2008). Subtype A consists of receptors for glycoprotein including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and chorionic gonadotropin (CG). All of which are dimeric proteins composed of a common α-subunit and a hormone-specific β-subunit (Van Loy et al. 2008). In mammals, subtype C refers to LGRs 7–8. This type of LGR receptors contains a unique N-terminal low-density lipoprotein (LDL) motif (Van Loy et al. 2008). Subtype B is composed of three mammalian receptors LGR4–6 characterized by the presence of 13–18 leucine-rich repeats (LRRs) (Van Loy et al. 2008). Many studies have confirmed that LGR5 and LGR6 are stem cell markers in several adult organs and tissues. Both of them are closely related to the carcinogenesis (Leushacke and Barker 2012).
Signaling Transduction of LGR4
The most well-known function of R-spondins-LGR4 pathway is to potentiate Wnt/β-catenin signaling. Overexpression of LGR4 significantly promotes the intracellular activity of Wnt signaling pathway induced by Rspo. Conversely, ablation of LGR4 completely abolishes Rspo-induced activity of β-catenin. Two mechanisms have been reported to mediate the action of Rspo-LGR4 on the potentiation of Wnt/β-catenin signaling. Firstly, Rspo-LGR4 functions to upregulate the level of Wnt receptors via direct suppression of membrane E3 ubiquitin ligases ZNRF3/RNF43. Alternatively, Rspo-LGR4 interacts with intracellular scaffold protein IQ motif containing GTPase-activating protein 1(IQGAP1), leading to robust activation of both canonical and noncanonical Wnt signaling pathways.
In the absence of R-spondins, two transmembrane E3 ligases RNF43/ZNRF3 interact with Wnt receptor complex Frizzled/LRP and inhibit Wnt signaling through accelerating the internalization and ubiquitination of Wnt receptor complex. Upon binding to LGR4, the furin-1 domain on R-spondins would simultaneously interact with ectodomain of ZNRF3, leading to the membrane clearance of RNF43/ZNRF3 and subsequent potentiation of Wnt signaling. Notably, activation of Wnt/β-catenin signaling pathway initiates the expression of proteins encoded by Wnt target genes, including RNF43/ZNRF3, which constitutes a negative feedback loop (Li et al. 2015).
Apart from neutralizing RNF43/ZNRF3 directly, Rspo-LGR4 may also modulate Wnt activity through recruiting IQGAP1 into the Wnt signaling complex. On one hand, Rspo-LGR4-IQGAP1 complex potentiates β-catenin-dependent canonical Wnt signaling pathway by enhancing MEK1/2-induced phosphorylation of LRP5/6. On the other hand, Rspo-LGR4-IQGAP1 contributes to the activation of β-catenin-independent noncanonical Wnt signaling through affecting actin dynamics (Li et al. 2015).
Importantly, a recent study indicates that Rspo proteins are not the only class of ligands for LGR4. Vertebrate norrin orthologous to insect burs and pburs could also stimulates Wnt signaling via a LGR4-dependent mechanism. The action of norrin is exclusive because it is not capable of activating LGR5 and 6 (Deng et al. 2013).
LGR4 in Embryonic Development
Studies have suggested that LGR4 acts as a crucial mediator in embryonic development as its ablation in mouse leads to embryonic/neonatal lethality. Specifically, the correlation between LGR4 and embryonic development has been demonstrated in a variety of organs and tissues, such as the eye, kidney, skin, reproductive system, gastrointestinal tract, etc.
LGR4 in Eye Development
LGR4 allele homozygous deletion mice manifest a consistent eye-open at birth (EOB) phenotype. This defect is primarily caused by impairment of proliferation and motility of keratinocytes companied by reduced F-actin in the eye epidermis (Wang et al. 2010). Lacking of LGR4 in cultured keratinocytes or developing eyelids strikingly blunts the phosphorylation of EGFR. Consistently, AG1478, an inhibitor of EGFR tyrosine kinase, restrains LGR4-dependent keratinocyte proliferation and motility (Wang et al. 2010). Previous study has identified HB-EGF as a core regulator responsible for the phosphorylation of EGFR, ERK, and STAT3. HB-EGF is diminished in Lgr4 (−/−) cell culture medium, whereas exogenous HB-EGF restores the activation of EGFR, ERK, and STAT3, as well as cell proliferation. Treatment of Lgr4 (+/+) keratinocytes with HB-EGF inhibitor CRM197 significantly blocks LGR4-mediated activation of EGFR (Wang et al. 2010). Taken together, LGR4 mediates EGFR signaling through HB-EGF and subsequently facilitates proliferation and motility of keratinocytes during embryonic eye development.
Other studies based on Lgr4 mutant mice report a correlation between LGR4 and age-related cataract. Phenotypically, Lgr4 (−/−) mice demonstrate earlier onset of lens opacification and higher susceptibility to cataract formation (Zhu et al. 2015). Evidenced by real-time quantitative PCR, two predominant antioxidant defense enzymes, catalase (CAT) and superoxidase dismutase-1 (SOD1), are significantly suppressed in the lens epithelial cells of Lgr4 (−/−) mice. These alterations may account for the early occurrence of cataract (Zhu et al. 2015).
LGR4 in Kidney Development
At E16.5, Lgr4-knockout mice display dilated kidney tubules and ectatic Bowman’s spaces. Consistent with this observation, mutation of Lgr4 results in earlier-arrested branching morphogenesis in the kidney at E15.5. Immunohistochemical analysis of Aqp3 reveals the premature differentiation of ureteric bud (UB) in Lgr4-defective kidney (Kinzel et al. 2014). Specifically, impaired UB differentiation caused by LGR4 ablation is due to the inactivation of Wnt pathway and its downstream effector Gata3 (Kinzel et al. 2014).
LGR4 in Skin Development
Skin dysplasia resulting from LGR4 deficiency occurs as early as E16.5, characterized by reduced basal cell proliferation and hair follicle numbers in the developing skin (Mohri et al. 2008). Consistent with this phenotype, another research has validated that genes essential for hair follicle morphogenesis, such as Edar, Lef1, and Shh, are prominently suppressed in LGR4-deficient mice. Correspondingly, the phosphorylation of Smad1/5/8 is potently strengthened. The increment in Smad1/5/8 phosphorylation disrupts normal follicle induction (Mohri et al. 2008).
LGR4 in Reproductive System Development
LGR4 knockout mice are infertile, indicating the pivotal role of LGR4 in the reproductive system. The ubiquitous presence of LGR4 during early prostate development suggests that LGR4 is likely to be involved in prostate morphogenesis. In support of this hypothesis, Lgr4 (−/−) mice demonstrate decreased prostate size along with aberrant luminal cell function. In vitro study in Lgr4 (−/−) Lin (−)/Sca1 (+)/CD49f (+) prostate stem cells (PSCs) has also confirmed that LGR4 ablation would impede PSC from differentiation. Similarly, in vivo kidney capsule prostate grafts lacking LGR4 expression demonstrate arrested PSC properties. Further investigation on the signaling mechanism reveals a dramatic decrease in Wnt, Notch1, and Sonic hedgehog expression in Lgr4 (−/−) cell. Remarkably, treatment with exogenous Sonic hedgehog partially reverses impaired PSCs differentiation caused by LGR4 ablation (Luo et al. 2013). All these observations suggest that LGR4 is capable of coordinating early prostate development and stem cell differentiation by regulating Wnt, Notch, and Sonic hedgehog signaling pathways.
During embryonic period, the development of female reproductive system, especially ovary and mammary glands, is also delicately modulated by LGR4. Lgr4 (−/−) female mice display abnormal development of Wolffian ducts and somatic cells which eventually develop into a phenotype of masculinization. In alignment with the loss of LGR4, target genes of Wnt/β-catenin signaling, such as lymphoid enhancer-binding factor 1 (Lef1) and Axin2 (Axin2), are downregulated (Koizumi et al. 2015). Furthermore, a variety of disruptions in mammary gland morphogenesis have been reported in Lgr4 (−/−) female mice, including postponed ductal development, decreased terminal end buds, and lessened side branching. Importantly, the potency of mammary stem cell repopulation is also severely affected (Wang et al. 2013). Again, the underlying mechanism mediating the effect of LGR4 on mammary development and stem cell function is Wnt/β-catenin/Lef1/Sox2 dependent (Wang et al. 2013).
LGR4 in the Development of Other Organs and Tissues
Apart from those mentioned above, LGR4 is also implicated in many other physiological activities, including myogenic differentiation, osteogenesis, fetal liver development, gall bladder and cystic duct development, innate immunity, cerebellar motor coordination, and synaptic plasticity, as well as the formation of cytonemes.
LGR4 in Gastrointestinal Homeostasis
Analyses of tissue distribution of LGR4 in the gastrointestinal tract reveal LGR4 in Paneth cells and crypt stem cells sandwiched between Paneth cells in a vesicular pattern (Yi et al. 2013). In a model of chemotherapy-induced intestinal mucositis, R-spondin1, the endogenous ligand of LGR4, promotes crypt cell proliferation (Li et al. 2015). This finding indicates the potential involvement of LGR4 in the maintenance of intestinal homeostasis. Compared to wild-type mice, deletion of LGR4 results in remarkable decrease in epithelial cell proliferation in postnatal mice. This change is associated with an 80% reduction in terminal differentiation of Paneth cells in intestinal crypts (Li et al. 2015). Using dextran sulfate sodium (DSS), Liu et al. have constructed an intestinal inflammation model and reported that LGR4 deficient mice exhibit significant reduced numbers of either Paneth cells or stem cells in the intestine. As a result, during the intestinal regeneration process, cell proliferation in Lgr4 (−/−) mice is severely impaired, and Lgr4 (−/−) mice demonstrate increased susceptibility and severity to DSS-induced inflammatory bowel disease. Further, reactivating Wnt/β-catenin signaling pathway protects Lgr4 (−/−) mice against inflammatory bowel disease (Li et al. 2015).
LGR4 in Energy Homeostasis
The immunoreactivity of LGR4 and its endogenous ligand Rspos can be detected in several hypothalamic nuclei critical for feeding behavior and energy metabolism, indicating the potential impact of Rspos-LGR4 signaling pathway on energy homeostasis. Studies using in situ hybridization have confirmed the expression of LGR4 in the arcuate nucleus (ARC), ventromedial nucleus of the hypothalamus (VMH), and median eminence of the hypothalamus. Rspo1 and Rspo3 are primarily located in VMH and paraventricular nucleus, respectively (Li et al. 2015). Fasting significantly suppresses the expression of both Rspo1 and Rspo3 in hypothalamus. Administration of Rspo1 or Rspo3 into the third brain ventricle induces an anorectic response. Conversely, expression of Rspo1 and Rspo3 is upregulated by insulin (Li et al. 2015). The anorexigenic effect of Rspo1 is at least partially due to the decrease of neuropeptide Y and increase of proopiomelanocortin expression in the ARC (Li et al. 2015). In peripheral tissues, LGR4 deficiency upregulates lipid oxidation-related gene expression while suppresses glucose transporter type 4 (Glut4) levels in skeletal muscle under fasting condition. These changes facilitate the transformation of primary energy substrate from glucose to fatty acid in response to energy depletion. Specifically, LGR4 dysfunction enhances energy adaptation in skeletal muscle by activating AMPK-SIRT1/PGC1α pathway (Sun et al. 2015).
As a novel obese-associated gene, heterozygous mutation of LGR4 correlates with decreased body weight in human. Lgr4 homozygous mutant (Lgr4 (m/m)) mice exhibit resistance to dietary and leptin mutant-induced obesity along with improved glucose metabolism (Li et al. 2015). In concert with this phenotype, LGR4 ablation potently potentiates browning of white adipose tissue (WAT), leading to increased energy consumption. Moreover, LGR4 may participate in coordinating plasma lipid rhythms. Wang et al. have discovered that the expression pattern of LGR4 in liver presents a rhythmic circadian fluctuation. Lack of LGR4, however, abrogates the diurnal expression pattern of the microsomal triglyceride transfer protein gene (Mttp) without altering other classic clock genes such as Clock, Baml1, Pers, etc. These observations may partially explain the disrupted plasma triglyceride rhythms observed in Lgr4 (m/m) mice. Although plasma cholesterol rhythm is not notably interfered by LGR4 dysfunction, Lgr4 (m/m) mice demonstrate significantly decreased levels of cholesterol (Li et al. 2015).
LGR4 in Human Diseases
LGR4 is critical for embryonic and postnatal development. Therefore, defected expression Lgr4 gene is closely related to a series of diseases, such as anterior segment dysgenesis (ASD), cataract, glaucoma, pseudohypoaldosteronism type 1 (PHA1), colorectal cancer, inflammatory bowel disease, etc. In addition to what have been discussed above, the role of LGR4 in AGR syndrome, oncogenesis, infertility, and osteoporosis has also been confirmed.
AGR syndrome, a genetic disorder caused by a contiguous gene deletion in the 11p13–14 region, is characterized by aniridia, genitourinary anomalies, and mental retardation. Interestingly, Yi et al. have determined LGR4 as the only G-protein-coupled receptor gene in human chromosome 11p12-11p14.4 fragment. This finding suggests that its mutation is likely to be responsible for the pathogenesis of AGR syndrome. As expected, knockdown of LGR4 in mouse leads to a variety of dysplasia similar to those observed in AGR syndrome. Specifically, LGR4 ablation attenuates the expression of histone demethylases Jmjd2a and Fbxl10 by targeting the cAMP-CREB signaling pathway (Yi et al. 2014).
Early in 2006, Gao et al. utilized microarray to screen genes differentially expressed in human colon carcinoma cells and identified Lgr4 as a candidate gene (Gao et al. 2006). In primary colorectal cancer (CRC) and metastatic lymph nodes, LGR4 is abundantly expressed. Further study indicates that abnormal expression of LGR4 in CRC evokes transcription activity of Wnt target gene T-cell factor 4 (TCF4) and the expression of TCF4-regulated genes including Cyclin D1 and c-Myc in CRC cells. These alterations consequently prompt the proliferation of CRC cells. Additionally, the effect of LGR4 on β-catenin/TCF signaling pathway is mediated by PI3K/Akt and mitogen-activated protein kinase/ERK1/2 pathway. Suppression of either pathway is sufficient to abolish the effect of LGR4 on oncogenesis (Wu et al. 2013). 5-Fluorouracil (5-FU) is widely used in chemotherapy as an effective treatment of colorectal cancer. Importantly, a novel research have identified that 5-FU induces apoptosis of colorectal cancer cells and inhibits proliferation by suppressing LGR4, indicating that LGR4 is a promising target for the treatment of colorectal cancer (Li et al. 2015). LGR4 activity is substantially upregulated in glioma tissues along with the activation of Wnt/β-catenin pathway. Induction of LGR4 expression accelerates the proliferation of glioma cells. On the other hand, challenging the glioma cells with small interfering RNA oligos targeting LGR4 negatively regulates proliferation activity of glioma cells (Yu et al. 2013). Taken together, all these observations affirm the role of LGR4 in oncogenesis.
Enhanced expression of LGR4 promotes migration and invasion of cancer cells and tumor metastasis in vitro and in vivo. A positive correlation has been demonstrated between the expression level of LGR4 and lymph node metastasis in human colon carcinoma (Gao et al. 2006). Overexpression of Lgr4 potentiates invasion and pulmonary metastasis capability of HCT116 human colon carcinoma cells. On the other hand, inactivation of LGR4 abolishes the invasive potential of HeLa and Lewis lung carcinoma cells (Gao et al. 2006). Thus, LGR4 is important in modulating invasiveness and metastasis of carcinoma.
In 2010, Mohri et al. first suggested the implication between LGR4 and the function of female reproductive system. Since then, a series of relevant researches has shed light on the specific roles of LGR4 in the regulation of fertility. The regulatory effect of progesterone on endometrial receptivity relies on LGR4 activity. In the context of LGR4 defection, the progesterone signaling in the uterus is significantly suppressed. Also abolished is the progesterone induced inhibition to luminal epithelial cell proliferation. Consistent with the alternation of ovarian hormone signaling, LGR4 defect female mice display uterine gland dysplasia, restrained stromal decidualization and subfertility (Kida et al. 2014). Additionally, LGR4 may play a vital role in mediating corpus luteum maturation. LGR4 activates EGFR-ERK signaling pathway in a Wnt/β-catenin-dependent manner to accelerate the maturation of luteum, maintaining the ability of female reproduction. LGR4-deficiencydramatically decreases serum levels of progesterone and prolongs estrous cycle in female mice. LGR4 knockdown eventually results in female infertility (Pan et al. 2014).
Similar consequences of LGR4 ablation have also been observed in male mice. LGR4 deficiency is closely associated with postnatal aberrant development of male reproductive system. Lgr4 homozygous mutant male mice undergo testis luminal swelling and rete testis dilation, defective elongation, and convolution of the efferent ducts and epididymis (Hoshii et al. 2007). Lgr4-deficient mice also demonstrate lack of estrogen receptor (ESR1) and SLC9A3 in the efferent ducts (Hoshii et al. 2007). Dysregulation of gene expression, together with shrunken epithelial area involved in liquid reabsorption, results in water reabsorption failure and luminal swelling. Taken together, LGR4 acts as a critical signaling molecule in the morphogenesis of testis and epididymis. Inactivation of LGR4 is correlated with decreased male fertility.
Other Human Diseases
A recent survey based on whole genome sequencing of Icelandic individuals expands the spectrum of LGR4 mutation-related human disorders. A rare nonsense mutation of LGR4 (c.376C>T) completely abolishes its function by terminating Lgr4 gene transcription at position 126 (Styrkarsdottir et al. 2013). The c.376C>T mutation of LGR4 exhibits a strong relevance to low bone mineral density (BMD) and osteoporotic fractures. Further, this mutation may be implicated in the pathogenesis of electrolyte imbalance, late onset of menarche, and reduced testosterone levels. Notably, the carriers of the c.376C>T mutation bear an increased susceptibility to squamous cell carcinoma of the skin and biliary tract cancer.
LGR4, a G-protein-coupled receptor, functions to potentiate both canonical and noncanonical Wnt signaling pathways upon activation by its ligands: R-spondins. As a critical molecular extensively expressed during embryonic development, LGR4 has been proved to mediate the morphogenesis of multiple organs and tissues, such as the gastrointestinal tract, eye, kidney, skin, reproductive system, etc. Correspondingly, dysfunction of LGR4 is responsible for several congenital dysplasias, including AGR syndrome, infertility, and impaired osteogenesis. Moreover, LGR4 may also play an important role in modulating energy homeostasis. Emerging evidences have also identified LGR4 as a potential poor prognostic factor in several human cancers. Overexpression of LGR4 in carcinoma is positively correlated with enhanced tumor invasiveness and metastasis. Therefore, further investigations focused on the functions and signaling mechanisms of LGR4 may provide novel therapeutic strategy for human diseases.
This work was supported by grants from the National Natural Science Foundation of China (81330010, 81390354) and American Diabetes Association grant #1-13-BS-225.
- Li Z, Zhang W, Mulholland MW. LGR4 and its role in intestinal protection and energy metabolism. Front Endocrinol. 2015;6(Lausanne):131. doi:10.3389/fendo.2015.00131.Google Scholar
- Zhu J, Hou Q, Dong XD, Wang Z, Chen X, Zheng D, et al. Targeted deletion of the murine Lgr4 gene decreases lens epithelial cell resistance to oxidative stress and induces age-related cataract formation. PLoS One. 2015;10(3):e0119599. doi:10.1371/journal.pone.0119599.PubMedPubMedCentralCrossRefGoogle Scholar