Leucine-rich repeat-containing, G protein-coupled receptor 4 null mice exhibit intrauterine growth retardation associated with embryonic and perinatal lethality

Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) belong to the largest mammalian superfamily of proteins with seven-transmembrane domains. LGRs can be divided into three subgroups based on their unique domain arrangement. Although two subgroups have been found to be receptors for glycoprotein hormones and relaxin-related ligands, respectively, the third LGR subgroup, consisting of LGR4-6, are orphan receptors with unknown physiological roles. To elucidate the functions of this subgroup of LGRs, LGR4 null mice were generated using a secretory trap approach to delete the majority of the LGR4 gene after the insertion of a beta-galactosidase reporter gene immediately after exon 1. Tissues expressing LGR4 were analyzed based on histochemical staining of the transgene driven by the endogenous LGR4 promoter. LGR4 was widely expressed in kidney, adrenal gland, stomach, intestine, heart, bone/cartilage, and other tissues. The expression of LGR4 in these tissues was further confirmed by immunohistochemical studies in wild-type animals. Analysis of the viability of 250 newborn animals suggested a skewed inheritance pattern, indicating that only 40% of the expected LGR4 null mice were born. For the LGR4 null mice viable at birth, most of them died within 2 d. Furthermore, the LGR4 null mice showed intrauterine growth retardation as reflected by a 14% decrease in body weight at birth, together with 30% and 40% decreases in kidney and liver weights, respectively. The present findings demonstrate the widespread expression of LGR4, and an essential role of LGR4 for embryonic growth, as well as kidney and liver development. The observed pre- and postnatal lethality of LGR4 null mice illustrates the importance of the LGR4 signaling system for the survival and growth of animals during the perinatal stage.     

Loss of Lgr4 inhibits differentiation,migration and apoptosis,and promotes proliferation in bone mesenchymal stem cells

The key signaling networks regulating bone marrow mesenchymal stem cells (BMSCs) are poorly defined. Lgr4, which belongs to the leucine-rich repeat-containing G protein-coupled receptor (LGR) family, is widely expressed in multiple tissues from early embryogenesis to adulthood. We investigated whether Lgr4 functions in BMSCs and in osteogenesis, adipogenesis, and skeletal myoblasts, using mice with a β-geo gene trap inserted into the Lgr4 gene. Abundant Lgr4 expression was detected in skeletal, adipose and muscular tissue of Lgr4^+/– mice at E16.5 by β-gal staining, and Lgr4-deficiency promoted BMSC proliferation (16 ± 4 in wild-type [WT] and 28 ± 2 in Lgr4^−/−) using colony forming units-fibroblast assay, while suppressing BMSC migration (from 103 ± 18 in WT to 57 ± 10 in Lgr4^−/−) by transwell migration assay and apoptosis ratio (from 0.0720 ± 0.0123 to 0.0189 ± 0.0051) by annexin V staining assay. Deletion of Lgr4 decreased bone mass (BV/TV from 19.16 ± 2.14 in WT mice to 10.36 ± 1.96 in KO) and fat mass through inhibiting BMSC differentiation to osteoblasts or adipocytes. Furthermore, LGR4-regulated osteogenic, adipogenic, and myogenic gene expression. Importantly, our data showed that loss of Lgr4-inhibited fracture healing by suppressing osteoblast differentiation. Moreover, deletion of Lgr4 in BMSCs-delayed fracture healing following stem cell therapy by BMSC transplantation. Together, our results demonstrated that LGR4 is essential for mesoderm-derived tissue development and BMSC differentiation, demonstrating that LGR4 could be a promising drug target for related diseases and a critical protein for stem cell therapy.     

Relaxin-3: improved synthesis strategy and demonstration of its high-affinity interaction with the relaxin receptor LGR7 both in vitro and in vivo

Relaxin-3 is a member of the human relaxin peptide family, the gene for which, RLN3, is predominantly expressed in the brain. Mapping studies in the rodent indicate a highly developed network of RLN3, RLN1, and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides have important functional roles in the central nervous system. A regioselective disulfide-bond synthesis protocol was developed and used for the chemical synthesis of human (H3) relaxin-3. The selectively S-protected A and B chains were combined by stepwise formation of each of the three insulin-like disulfides via aeration, thioloysis, and iodolysis. Judicious positioning of the three sets of S-protecting groups was crucial for acquisition of synthetic H3 relaxin in a good overall yield. The activity of the peptide was tested against relaxin family peptide receptors. Although the highest activity was demonstrated on the human relaxin-3 receptor (GPCR135), the peptide also showed high activity on relaxin receptors (LGR7) from various species and variable activity on the INSL3 receptor (LGR8). Recombinant mouse prorelaxin-3 demonstrated similar activity to H3 relaxin, suggesting that the presence of the C peptide did not influence the conformation of the active site. H3 relaxin was also able to activate native LGR7 receptors. It stimulated increased MMP-2 expression in LGR7-expressing rat ventricular fibroblasts in a dose-dependent manner and, following infusion into the lateral ventricle of the brain, stimulated water drinking in rats, activating LGR7 receptors located in the subfornical organ. Thus, H3 relaxin is able to interact with the relaxin receptor LGR7 both in vitro and in vivo.     

Crystal Structure of LGR4-Rspo1 Complex: INSIGHTS INTO THE DIVERGENT MECHANISMS OF LIGAND RECOGNITION BY LEUCINE-RICH REPEAT G-PROTEIN-COUPLED RECEPTORS (LGRs)*

Leucine-rich repeat G-protein-coupled receptors (LGRs) are a unique class of G-protein-coupled receptors characterized by a large extracellular domain to recognize ligands and regulate many important developmental processes. Among the three groups of LGRs, group B members (LGR4–6) recognize R-spondin family proteins (Rspo1–4) to stimulate Wnt signaling. In this study, we successfully utilized the “hybrid leucine-rich repeat technique,” which fused LGR4 with the hagfish VLR protein, to obtain two recombinant human LGR4 proteins, LGR4₁₅ and LGR4₉. We determined the crystal structures of ligand-free LGR4₁₅ and the LGR4₉-Rspo1 complex. LGR4 exhibits a twisted horseshoe-like structure. Rspo1 adopts a flat and β-fold architecture and is bound in the concave surface of LGR4 in the complex through electrostatic and hydrophobic interactions. All the Rspo1-binding residues are conserved in LGR4–6, suggesting that LGR4–6 bind R-spondins through an identical surface. Structural analysis of our LGR4-Rspo1 complex with the previously determined LGR4 and LGR5 structures revealed that the concave surface of LGR4 is the sole binding site for R-spondins, suggesting a one-site binding model of LGR4–6 in ligand recognition. The molecular mechanism of LGR4–6 is distinct from the two-step mechanism of group A receptors LGR1–3 and the multiple-interface binding model of group C receptors LGR7–8, suggesting LGRs utilize the divergent mechanisms for ligand recognition. Our structures, together with previous reports, provide a comprehensive understanding of the ligand recognition by LGRs.     

LGR5 regulates osteogenic differentiation of human thoracic ligamentum flavum cells by Wnt signalling pathway

Thoracic ossification of the ligamentum flavum (TOLF) is ectopic ossification of the spinal ligaments. Histologically, the development of TOLF can be described as the process of endochondral ossification. However, the underlying aetiology has not been completely clarified. In this investigation, the gene expression profile associated with leucine‐rich repeat‐containing G‐protein‐coupled receptors (LGR) and Wnt signalling pathway in the thoracic ligamentum flavum cells (TLFCs) of different ossification stages was analysed via RNA sequencing. We further confirmed the significant differences in the related gene expression profile by Gene Ontology (GO) enrichment analysis. LGR5 was first identified in primary human TLFCs during osteogenic differentiation. To evaluate the effect of LGR5 on osteogenic differentiation, LGR5 has been knocked down and overexpressed in human TLFCs. We observed that the knockdown of LGR5 inhibited the activity of Wnt signalling and attenuated the potential osteogenic differentiation of TLFCs, while overexpression of LGR5 activated the Wnt signalling pathway and increased osteogenic differentiation. Our results provide important evidence for the potent positive mediatory effects of LGR5 on osteogenesis by enhancing the Wnt signalling pathway in TOLF.     

The three subfamilies of leucine-rich repeat-containing G protein-coupled receptors (LGR): identification of LGR6 and LGR7 and the signaling mechanism for LGR7

Glycoprotein hormone receptors, including LH receptor, FSH receptor, and TSH receptor, belong to the large G protein-coupled receptor (GPCR) superfamily but are unique in having a large ectodomain important for ligand binding. In addition to two recently isolated mammalian LGRs (leucine-rich repeat-containing, G protein-coupled receptors), LGR4 and LGR5, we further identified two new paralogs, LGR6 and LGR7, for glycoprotein hormone receptors. Phylogenetic analysis showed that there are three LGR subgroups: the known glycoprotein hormone receptors; LGR4 to 6; and a third subgroup represented by LGR7. LGR6 has a subgroup-specific hinge region after leucine-rich repeats whereas LGR7, like snail LGR, contains a low density lipoprotein (LDL) receptor cysteine-rich motif at the N terminus. Similar to LGR4 and LGR5, LGR6 and LGR7 mRNAs are expressed in multiple tissues. Although the putative ligands for LGR6 and LGR7 are unknown, studies on single amino acid mutants of LGR7, with a design based on known LH and TSH receptor gain-of-function mutations, indicated that the action of LGR7 is likely mediated by the protein kinase A but not the phospholipase C pathway. Thus, mutagenesis of conserved residues to allow constitutive receptor activation is a novel approach for the characterization of signaling pathways of selective orphan GPCRs. The present study also defines the existence of three subclasses of leucine-rich repeat-containing, G protein-coupled receptors in the human genome and allows future studies on the physiological importance of this expanding subgroup of GPCR.     

Establishment of a novel monoclonal antibody against LGR5

LGR5 is an orphan G-protein-coupled receptor (GPCR) that is expressed on the cell surface membrane. LGR5 is reported to be overexpressed in colon, liver, and ovary tumor compared to normal tissue. However, a specific ligand for LGR5 has not yet been determined, and the function is still not clear. An LGR5-specific monoclonal antibody (mAb) is needed as a tool for detection and analysis of LGR5 biological function and cancer therapy. To date, no mAb against LGR5 that retains high affinity and specificity has been reported. Here, we report successful establishment and characterization of a mAb (KM4056) that specifically recognizes the extracellular N-terminal domain of human LGR5, but not LGR4 or LGR6. This mAb has potent complement-dependent cytotoxicity (CDC) activity in vitro and shows strong anti-tumor activity in vivo against xenograft model by transplanting LGR5 expressing CHO transfectants into SCID mice. Thus, KM4056 can be a useful tool for detection of LGR5 positive cells and analysis of LGR5 biological function.     

LGR4 is required for sequential molar development

Tooth development requires proliferation, differentiation, and specific migration of dental epithelial cells, through well-organized signaling interactions with mesenchymal cells. Recently, it has been reported that leucine-rich repeat-containing G protein coupled receptor 4 (LGR4), the receptor of R-spondins, is expressed in many epithelial cells in various organs and tissues and is essential for organ development and stem cell maintenance. Here, we report that LGR4 contributes to the sequential development of molars in mice. LGR4 expression in dental epithelium was detected in SOX2⁺ cells in the posterior end of the second molar (M2) and the early tooth germ of the third molar (M3). In keratinocyte-specific Lgr4-deficient mice (Lgr4^{K5 KO}), the developmental defect became obvious by postnatal day 14 (P14) in M3. Lgr4^{K5 KO} adult mice showed complete absence or the dwarfed form of M3. In M3 development in Lgr4^{K5 KO} mice, at Wnt/β-catenin signal activity was down-regulated in the dental epithelium at P3, as indicated by lymphoid enhancer-binding factor-1 (LEF1) expression. We also confirmed the decrease, in dental epithelium of Lgr4^{K5 KO} mice, of the number of SOX2⁺ cells and the arrest of cell proliferation at P7, and observed abnormal differentiation at P14. Our data demonstrated that LGR4 controls the sequential development of molars by maintaining SOX2⁺ cells in the dental epithelium, which have the ability to form normal molars.     

The relaxin peptide family and their novel G-protein coupled receptors

Relaxin-1 is a heterodimeric peptide hormone primarily produced by the pregnant corpus luteum and/or placenta and is involved in many essential physiological processes centered on its action as a potent extracellular matrix (ECM) remodeling agent. Insulin-like peptide 3 (INSL3), also known as relaxin-like factor, is predominantly expressed in the Leydig cells of the testes and is an important mediator of testicular descent. The relaxin-1 equivalent peptide in humans is actually the product of the human RLN2 gene, human 2 (H2) relaxin. Recently identified and thought to be the ancestral relaxin, relaxin-3 is specifically expressed in the nucleus incertus of the mouse and rat brain and is most likely an important neuropeptide. Each of the hormones above act on cell membrane G-protein coupled receptors (GPCRs). The relaxin-1 receptor is leucine-rich repeat-containing GPCR 7 (LGR7) whereas INSL3 acts on the closely related LGR8. These receptors have large extra-cellular domains containing multiple leucine-rich repeats (LRRs) and a unique LDL receptor-like cysteine-rich motif (LDLR-domain). Relaxin-3 will bind and activate LGR7 with 50-fold lower activity than H2 relaxin. Two relaxin-3 selective GPCRs; somatostatin and angiotensin like peptide receptor (SALPR) and GPCR 142 were recently identified, these type I GPCRs are unrelated to LGR7 and LGR8. The discovery and characterisation of these receptors is greatly aiding the quest to unravel the mechanics of these important hormones, however with three other family members, insulin-like peptides 4–6 (INSL4, INSL5 and INSL6) with unknown functions and unidentified receptors, there is still much to be learnt about this hormone family.     

Analysis of LGR4 Receptor Distribution in Human and Mouse Tissues

LGR4 is an R-spondin receptor with strong positive effect on Wnt signaling. It plays a critical role in development as its ablation in the mouse led to total embryonic/neonatal lethality with profound defects in multiple organs. Haplotype insufficiency of LGR4 in human was associated with several diseases, including increased risk of squamous cell carcinoma of the skin, reduced birth weights, electrolyte imbalance, and decreased levels of testosterone, which are similar to the phenotypes of LGR4-hypomorphic mice. Tissue distribution of LGR4 was extensively analyzed in the mouse using gene-trap reporter enzyme alleles. However, its expression pattern in human tissues remained largely unknown. We have developed LGR4-specific monoclonal antibodies and used them to examine the expression of LGR4 in selected adult human and mouse tissues by immunohistochemical analysis. Intense LGR4-like immunoreactivity was observed in the epidermis and hair follicle of the skin, pancreatic islet cells, and epithelial cells in both the male and female reproductive organs. Of particular interest is that LGR4 is highly expressed in germ cells and pancreatic islet cells, which have important implications given the role of R-spondin-LGR4 signaling in the survival of adult stem cells. In addition, the majority of colon tumors showed elevated levels of LGR4 receptor. Overall, the expression pattern of LGR4 in human tissues mapped by this IHC analysis is similar to that in the mouse as revealed from gene trap alleles. Importantly, the pattern lends strong support to the important role of LGR4 in the development and maintenance of skin, kidney, reproductive systems, and other organs.     

LGR5/R-Spo1/Wnt3a axis promotes stemness and aggressive phenotype in hepatoblast-like hepatocellular carcinoma cell lines

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a newly defined stem cell marker in endoderm-derived organs such as the small intestine, colon and pancreas. Recently, LGR5 was demonstrated to be an important factor in liver regeneration and stem cell maintenance. Moreover, LGR5 expression is highly up-regulated in various cancers including hepatocellular carcinoma. Herein, we demonstrate that LGR5 expression is specifically observed in certain subset of HCC cell lines with “hepatoblast-like” appearance, characterized by the expression of liver fetal/progenitor markers. Notably, the activation of the canonical Wnt pathway significantly increases the expression of LGR5 in this subset of cell lines, whereas it does not cause any induction of LGR5 expression in mesenchymal like cell lines SNU-475 and SNU-449. Furthermore, we showed that treatment of the hepatoblast-like HCC cell lines HuH-7 and Hep3B with LGR5 ligand R-Spo1 significantly amplifies the induction of LGR5 expression, the phosphorylation of LRP6 and β-catenin resulting in enhanced TCF/LEF activity either alone or in combination with Wnt3a. Consistently, the silencing of the LGR5 gene attenuates the co-stimulatory effect of R-Spo1/Wnt3a on TCF/LEF activity while overexpression of LGR5 enhances it. On the contrary, overexpression of LGR5 does not change TCF/LEF activity induced by R-Spo1/Wnt3a in mesenchymal-like HCC line, SNU-449. Importantly, LGR5-overexpressing cells have increased expression of several Wnt target genes and stemness-related genes including EpCAM and CK19 upon R-Spo1/Wnt3a treatment. LGR5-overexpressing cells also have increased spheroid forming, migration and invasion abilities and stimulation with R-Spo1/Wnt3a augments these abilities of LGR5-overexpressing cells. In addition, ectopic overexpression of LGR5 significantly increases cell proliferation rate independent of R-Spo1/Wnt3a stimulation. Moreover, in vitro tubulogenesis assay demonstrates that treatment with R-Spo1/Wnt3a enhances the sprouting of capillary tubules in only LGR5-overexpressing cells. Finally, R-Spo1/Wnt3a significantly promotes dissemination of LGR5-overexpressing cells in vivo in a zebrafish xenograft model. Our study unravels a tumor-promoting role for LGR5 through activation of canonical Wnt/β-catenin signaling in the hepatoblast-like HCCs. In conclusion, our results suggest that LGR5/R-Spo1/Wnt3a generates an axis that mediates the acquisition of aggressive phenotype specifically in hepatoblast-like subset of HCCs and might represent a valuable target for treatment of HCC tumors with aberrant activation of Wnt/β-catenin pathway.     

Human placental alkaline phosphatase as a histochemical marker of gene expression in transgenic mice

The human placental alkaline phosphatase (PLAP) gene was analysed for its utility as a histochemically detectable reporter gene in transgenic mice. A reporter gene was made by linking the PLAP structural gene to an enhancerpromoter element from the human -actin gene. This gene was inserted into the mouse genome by transfection of embryonic stem cells, and by microinjection of fertilized eggs. Histochemical staining showed that the transgene was uniformly expressed in four of four stable ES cell lines, and in all ten tissues examined from adult animals from five lines of transgenic mice. Non-transgenic cells did not stain. These results suggest that the human PLAP gene will be of utility in studies requiring phenotypic marking of cells in tissues of mice.     

The nematode leucine-rich repeat-containing, G protein-coupled receptor (LGR) protein homologous to vertebrate gonadotropin and thyrotropin receptors is constitutively active in mammalian cells

The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane protein family and are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of an expanding family of homologous leucine-rich repeat-containing, G protein-coupled receptors (LGRs), including the three known glycoprotein hormone receptors; mammalian LGR4 and LGR5; and LGRs in sea anemone, fly, and snail. We isolated nematode LGR cDNA and characterized its gene from the Caenorhabditis elegans genome. This receptor cDNA encodes 929 amino acids consisting of a signal peptide for membrane insertion, an ectodomain with nine leucine-rich repeats, a seven-TM region, and a long C-terminal tail. The nematode LGR has five potential N-linked glycosylation sites in its ectodomain and multiple consensus phosphorylation sites for protein kinase A and C in the cytoplasmic loop and C tail. The nematode receptor gene has 13 exons; its TM region and C tail, unlike mammalian glycoprotein hormone receptors, are encoded by multiple exons. Sequence alignments showed that the TM region of the nematode receptor has 30% identity and 50% similarity to the same region in mammalian glycoprotein hormone receptors. Although human 293T cells expressing the nematode LGR protein do not respond to human glycoprotein hormones, these cells exhibited major increases in basal cAMP production in the absence of ligand stimulation, reaching levels comparable to those in cells expressing a constitutively activated mutant human LH receptor found in patients with familial male-limited precocious puberty. Analysis of cAMP production mediated by chimeric receptors further indicated that the ectodomain and TM region of the nematode LGR and human LH receptor are interchangeable and the TM region of the nematode LGR is responsible for constitutive receptor activation. Thus, the identification and characterization of the nematode receptor provides the basis for understanding the evolutionary relationship of diverse LGRs and for future analysis of mechanisms underlying the activation of glycoprotein hormone receptors and related LGRs.     

Transgenic mice ubiquitously expressing human placental alkaline phosphatase (PLAP): an additional reporter gene for use in tandem with beta-galactosidase (lacZ)

A fundamental keystone of developmental biology has been the growing use of reporter genes in model transgenic systems. Their use has greatly facilitated investigations of cell lineage and cell fate in addition to aiding experiments aimed at determining patterns of gene expression, gene interaction and gene regulation. Through construction of transgenic mice, ubiquitously expressing human placental alkaline phosphatase (PLAP), we demonstrate the suitability of PLAP as a reporter gene for use in conjunction with, or as an alternative to, beta-galactosidase (lacZ). Our findings demonstrate that over-expression of PLAP has no adverse effects on mouse development or viability, despite a widespread pattern of expression. This technology provides a simple yet effective mechanism based on eukaryotic reporter gene technology to facilitate the identification of transgenic cells within complex in vivo systems.