Jag2b-Notch3/1b-mediated neuron-to-glia crosstalk controls retinal gliogenesis.

In the developing central nervous systems (CNS), neural progenitor cells generate neurons and glia in sequential order. However, the influence of neurons on glia generation remains elusive. Here, we report that photoreceptor cell-derived Jag2b is required for Notch-dependent Müller glia (MG) generation in the developing zebrafish retina. In jab2b-/- mutants, differentiating MGs are re-specified into lineage-related bipolar neuron fate at the expense of mature MG. Single-cell transcriptome analysis and knock-in animals reveal that jab2b is specifically expressed in crx+ -photoreceptor cells during MG generation. Crx promoter-driven jag2b, but not other Notch ligands, is sufficient to rescue the loss of MGs observed in jag2b-/- mutants. Furthermore, we observe a severe and moderate decrease in the number of MGs in notch3-/- and notch1b-/- mutants, respectively, and the activation of Notch3 or Notch1b rescues the MG loss in jag2b-/- mutants. Together, our findings reveal that the interaction of Jag2b and Notch3/Notch1b mediates the crosstalk between neurons and glial cells to ensure the irreversible differentiation of MG, providing novel mechanistic insights into the temporal specification of glial cell fate in a developing vertebrate CNS structure.

Rigorous coupled-wave analysis of unilateral Smith-Purcell radiation from asymmetric resonators.

The Smith-Purcell radiation produced by electrons moving closely to a grating can be enhanced by resonances. Here, we show a method to manipulate the directionality of the resonance-enhanced radiation. Using the rigorous coupled-wave analysis method, we compare the radiation from symmetric and asymmetric gratings, showing that the enhanced Smith-Purcell radiation can become unilateral with a perturbation that breaks the structural symmetry. Our work provides an effective method for frequency-domain calculation of Smith-Purcell radiation and also an approach to realize more efficient use of the radiation.

Progressively Decreased HCN1 Channels Results in Cone Morphological Defects in Diabetic Retinopathy.

Historically, diabetic retinopathy has been recognized as a vascular disease. Recent clinical evidence suggests the initiation of diabetic retinopathy with neuropathy rather than microangiopathy. However, the molecular mechanism that drives diabetic retinopathy-associated neuropathy remains mostly unexplored. Here, we reported progressive diabetic retinopathy defects in blood glucose levels, shortening of cone segments and uncoupled appearance of retinal vascular abnormalities from pdx1 +/- mutants zebrafish to glucose-treated pdx1 +/- mutants zebrafish of both sexes. Further single-cell transcriptomic analysis revealed cones as the most vulnerable retinal neuron type that underwent three developmentally progressive cell states (States 1-3), predominantly present in WT animals, pdx1 +/- mutants, and glucose-treated pdx1 +/- mutants, respectively. Mechanistically, the expression of hcn1 was progressively decreased in cones during its transition from State 1 to State 3. Furthermore, genetic hcn1 disruption resulted in similar cone segment defects found in the diabetic retinopathy model, suggesting the involvement of progressive hcn1 reduction in diabetic retinopathy-associated cone defects. Thus, our study provided a vertebrate retina model representing progressive diabetic retinopathy defects and further gained new mechanistic insights into the cone morphologic defects as an early neuropathy in diabetic retinopathy.SIGNIFICANCE STATEMENT We create a vertebrate retina model representing the progressive diabetic retinopathy-associated defects using zebrafish. Further systematic single-cell transcriptome analysis reveals two novel cell states of cones in response to different levels of higher glucose and the progressive decrease of HCN1 channels as a mechanism underlying cone defects in diabetic retinopathy.

Unifying developmental programs for embryonic and postembryonic neurogenesis in the zebrafish retina.

The zebrafish retina grows for a lifetime. Whether embryonic and postembryonic retinogenesis conform to the same developmental program is an outstanding question that remains under debate. Using single-cell RNA sequencing of ∼20,000 cells of the developing zebrafish retina at four different stages, we identified seven distinct developmental states. Each state explicitly expresses a gene set. Disruption of individual state-specific marker genes results in various defects ranging from small eyes to the loss of distinct retinal cell types. Using a similar approach, we further characterized the developmental states of postembryonic retinal stem cells (RSCs) and their progeny in the ciliary marginal zone. Expression pattern analysis of state-specific marker genes showed that the developmental states of postembryonic RSCs largely recapitulated those of their embryonic counterparts, except for some differences in rod photoreceptor genesis. Thus, our findings reveal the unifying developmental program used by the embryonic and postembryonic retinogenesis in zebrafish.

Enhancement of Smith-Purcell radiation from cylindrical gratings by quasi-bound states in the continuum.

Smith-Purcell radiation (SPR) is an important means of generating terahertz waves, and the enhancement of SPR is an attractive topic nowadays. Inspired by the phenomenon of special SPR, where the enhancement is achieved by using a high-duty-cycle grating, we describe a new, to the best of our knowledge, but more effective approach to this challenging problem. By deriving a simple analytical solution for the SPR from an annular electron beam passing through a cylindrical metallic grating, we show that the inverse structure, a low-duty-cycle grating can exhibit rather high SPR efficiencies in the presence of quasi-bound states in the continuum (quasi-BICs). The analytical prediction is supported by particle-in-cell simulations, which show that the quasi-BICs can enhance the superradiant SPR generated by a train of electron bunches by orders of magnitude. These results present an interesting mechanism for enhancing the SPR from metallic gratings, and may find applications in terahertz free-electron lasers.

Sequence, structure, and function of the Dps DNA-binding protein from Deinococcus wulumuqiensis R12.

Deinococcus wulumuqiensis R12, which was isolated from arid irradiated soil in Xinjiang province of China, belongs to a genus that is well-known for its extreme resistance to ionizing radiation and oxidative stress. The DNA-binding protein Dps has been studied for its great contribution to oxidative resistance. To explore the role of Dps in D. wulumuqiensis R12, the Dps sequence and homology-modeled structure were analyzed. In addition, the dps gene was knocked out and proteomics was used to verify the functions of Dps in D. wulumuqiensis R12. Docking data and DNA binding experiments in vitro showed that the R12 Dps protein has a better DNA binding ability than the Dps1 protein from D. radiodurans R1. When the dps gene was deleted in D. wulumuqiensis R12, its resistance to H2O2 and UV rays was greatly reduced, and the cell envelope was destroyed by H2O2 treatment. Additionally, the qRT-PCR and proteomics data suggested that when the dps gene was deleted, the catalase gene was significantly down-regulated. The proteomics data indicated that the metabolism, transport and oxidation-reduction processes of D. wulumuqiensis R12 were down-regulated after the deletion of the dps gene. Overall, the data conformed that Dps protein plays an important role in D. wulumuqiensis R12.

All-trans retinoic acid impairs glucose-stimulated insulin secretion by activating the RXR/SREBP-1c/UCP2 pathway.

Diabetes is often associated with vitamin A disorders. All-trans retinoic acid (ATRA) is the main active constituent of vitamin A. We aimed to investigate whether ATRA influences diabetic progression and its mechanisms using both Goto-Kazizazi (GK) rats and INS-1 cells. Rat experiments demonstrated that ATRA treatment worsened diabetes symptoms, as evidenced by an increase in fasting blood glucose (FBG) levels and impairment of glucose homeostasis. Importantly, ATRA impaired glucose-stimulated insulin secretion (GSIS) and increased the expression of sterol regulatory element-binding protein 1c (SREBP-1c) and uncoupling protein 2 (UCP2) in the rat pancreas. Data from INS-1 cells also showed that ATRA upregulated SREBP-1c and UCP2 expression and impaired GSIS at 23 mM glucose. Srebp-1c or Ucp2 silencing attenuated GSIS impairment by reversing the ATRA-induced increase in UCP2 expression and decrease in ATP content. ATRA and the retinoid X receptor (RXR) agonists 9-cis RA and LG100268 induced the gene expression of Srebp-1c, which was almost completely abolished by the RXR antagonist HX531. RXRα-LBD luciferase reporter plasmid experiments also demonstrated that ATRA concentration-dependently activated RXRα, the EC50 of which was 1.37 µM, which was lower than the ATRA concentration in the pancreas of GK rats treated with a high dose of ATRA (approximately 3 µM), inferring that ATRA can upregulate Srebp-1c expression in the pancreas by activating RXR. In conclusion, ATRA impaired GSIS partly by activating the RXR/SREBP-1c/UCP2 pathway, thus worsening diabetic symptoms. The results highlight the roles of ATRA in diabetic progression and establish new strategies for diabetes treatment.

RNA editing enzyme adenosine deaminases acting on RNA 1 deficiency increases the sensitivity of non-small cell lung cancer cells to anlotinib by regulating CX3CR1-fractalkine expression.

Adenosine deaminases acting on RNA 1 (ADAR1) has been identified to play key roles in non-small cell lung cancer (NSCLC) progression, and can modulate the sensitivity of cancer cells to anticancer drugs. The current study aimed to investigate the effect of ADAR1 on the sensitivity of NSCLC cells to anlotinib. We established anlotinib-resistant NSCLC (NSCLC/AR) cells, including NCI-H1975/AR and A549/AR cells. Results showed that ADAR1 was significantly upregulated in NSCLC/AR cells. Genetic-knockdown of ADAR1 increased the sensitivity of NSCLC/AR cells to anlotinib by inducing cell proliferation suppression, cell cycle arrest, and apoptosis. Furthermore, knockdown of ADAR1 decreased the level of C-X3-C motif chemokine ligand 1 (CX3CL1) in NCI-H1975/AR and A549/AR cells after anlotinib treatment. Introduction of exogenous CX3CL1 significantly reversed the positive effect of ADAR1 deficiency on NSCLC/AR cell sensitivity, exhibited by the increase of cell viability and decrease of apoptosis. Further in-vivo study demonstrated that knockdown of ADAR1 inhibited NCI-H1975/AR cell tumorigenesis by reducing CX3CL1 expression. Collectively, ADAR1 deficiency increased the sensitivity of NSCLC/AR cells to anlotinib by downregulating CX3CL1, which might provide a potential strategy for NSCLC/AR therapy.

A Site Distance Effect Induced by Reactant Molecule Matchup in Single-Atom Catalysts for Fenton-Like Reactions.

Understanding the site interaction nature of single-atom catalysts (SACs), especially densely populated SACs, is vital for their application to various catalytic reactions. Herein, we report a site distance effect, which emphasizes how well the distance of the adjacent copper atoms (denoted as dCu1-Cu1 ) matches with the reactant peroxydisulfate (PDS) molecular size to determine the Fenton-like reaction reactivity on the carbon-supported SACs. The optimized dCu1-Cu1 in the range of 5-6 Å, which matches the molecular size of PDS, endows the catalyst with a nearly two times higher turnover frequency than that of dCu1-Cu1 beyond this range, accordingly achieving record-breaking kinetics for the oxidation of emerging organic contaminants. Further studies suggest that this site distance effect originates from the alteration of PDS adsorption to a dual-site structure on Cu1 -Cu1 sites when dCu1-Cu1 falls within 5-6 Å, significantly enhancing the interfacial charge transfer and consequently resulting in the most efficient catalyst for PDS activation so far.

Bile duct ligation causes opposite impacts on the expression and function of BCRP and P-gp in rat brain partly via affecting membrane expression of ezrin/radixin/moesin proteins.

Breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) are co-located at blood-brain barrier (BBB) cells, preventing their substrates from entering brain. Accumulating evidence demonstrates that liver failure impairs P-gp and BCRP expression and function in the brain. In the current study, we investigated how liver failure influenced the expression and function of brain BCRP and P-gp in rats subjected to bile duct ligation (BDL). The function of BCRP, P-gp and BBB integrity was assessed using distribution of prazosin, rhodamine 123 and fluorescein, respectively. We showed that BDL significantly decreased BCRP function, but increased P-gp function without affecting BBB integrity. Furthermore, we found that BDL significantly downregulated the expression of membrane BCRP and upregulated the expression of membrane P-gp protein in the cortex and hippocampus. In human cerebral microvascular endothelial cells, NH4Cl plus unconjugated bilirubin significantly decreased BCRP function and expression of membrane BCRP protein, but upregulated P-gp function and expression of membrane P-gp protein. The decreased expression of membrane BCRP protein was linked to the decreased expression of membrane radixin protein, while the increased expression of membrane P-gp protein was related to the increased location of membrane ezrin protein. Silencing ezrin impaired membrane location of P-gp, whereas silencing radixin impaired membrane location of BCRP protein. BDL rats showed the increased expression of membrane ezrin protein and decreased expression of membrane radixin protein in the brain. We conclude that BDL causes opposite effects on the expression and function of brain BCRP and P-gp, attributing to the altered expression of membrane radixin and ezrin protein, respectively, due to hyperbilirubinemia and hyperammonemia.

Short-chain fatty acids exert opposite effects on the expression and function of p-glycoprotein and breast cancer resistance protein in rat intestine.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are involved in intestinal barrier. Short-chain fatty acids (SCFAs) play important roles in maintaining intestinal barrier. In this study we explored how SCFAs affected the expression and function of intestinal P-gp and BCRP in rats. Rats received 150 mM acetate, propionate or butyrate in drinking water for 4 weeks. In SCFA-treated rats, the expression and function of intestinal P-gp were decreased, but those of intestinal BCRP were increased; intestinal p-p65 was also decreased, which was positively related to P-gp protein expression. Among the three SCFAs tested, butyrate exhibited the strongest induction or inhibitory effect, followed by propionate and acetate. Similar results were observed in mouse primary enterocytes and Caco-2 cells treated with acetate (5 mM), propionate (2 mM), or butyrate (1 mM). In Caco-2 cells, addition of butyrate, vorinostat, and valproate (two classic HDAC inhibitors), Bay117082 (selective inhibitor of NF-κB activation) or NF-κB p65 silencing significantly decreased the expression of P-gp and the level of phosphorylated p65 (p-p65). Furthermore, butyrate attenuated the expression of P-gp and p-p65 induced by TNF-α (NF-κB activator) and theophylline (HDAC activator). However, vorinostat, valproate, Bay117082, TNF-α or p65 silencing hardly affected BCRP protein expression. But GW9662 (selective PPARγ antagonist) or PPARγ silencing abolished BCRP induction by butyrate and troglitazone (PPARγ agonist). SCFAs-treated rats showed higher intestinal protein expression of PPARγ, which was positively related to BCRP protein expression. Butyrate increased plasma exposure of fexofenadine but decreased that of rosuvastatin following oral dose to rats. In conclusion, SCFAs exert opposite effects on the expression and function of intestinal P-gp and BCRP; butyrate downregulated P-gp expression and function possibly via inhibiting HDAC/NF-κB pathways; butyrate induced BCRP expression and function partly via PPARγ activation.

Interleukin-17 and Th17 Lymphocytes Directly Impair Motoneuron Survival of Wildtype and FUS-ALS Mutant Human iPSCs.

Amyotrophic lateral sclerosis (ALS) is a progressive disease leading to the degeneration of motor neurons (MNs). Neuroinflammation is involved in the pathogenesis of ALS; however, interactions of specific immune cell types and MNs are not well studied. We recently found a shift toward T helper (Th)1/Th17 cell-mediated, pro-inflammatory immune responses in the peripheral immune system of ALS patients, which positively correlated with disease severity and progression. Whether Th17 cells or their central mediator, Interleukin-17 (IL-17), directly affects human motor neuron survival is currently unknown. Here, we evaluated the contribution of Th17 cells and IL-17 on MN degeneration using the co-culture of iPSC-derived MNs of fused in sarcoma (FUS)-ALS patients and isogenic controls with Th17 lymphocytes derived from ALS patients, healthy controls, and multiple sclerosis (MS) patients (positive control). Only Th17 cells from MS patients induced severe MN degeneration in FUS-ALS as well as in wildtype MNs. Their main effector, IL-17A, yielded in a dose-dependent decline of the viability and neurite length of MNs. Surprisingly, IL-17F did not influence MNs. Importantly, neutralizing IL-17A and anti-IL-17 receptor A treatment reverted all effects of IL-17A. Our results offer compelling evidence that Th17 cells and IL-17A do directly contribute to MN degeneration.

Circular RNA sequencing indicates circ-IQGAP2 and circ-ZC3H6 as noninvasive biomarkers of primary Sjögren's syndrome.

OBJECTIVES: This study aims to characterize the expression profiles of circRNAs in primary Sjogren's Syndrome (pSS) and examine the potential of noninvasive circular RNAs (circRNAs) as biomarkers of pSS. METHODS: We performed RNA sequencing of minor salivary gland (MSG) biopsies from four pSS and four non-pSS individuals (subjects undergoing MSG biopsies but not meeting 2012 or 2016 ACR classification criteria for SS). Differentially expressed circRNAs were identified by DESeq2, and confirmed by quantitative real-time PCR in the MSGs as well as in plasma exosomes in 37 pSS and 14 non-pSS subjects. Discriminatory capacity testing using receiver operating characteristic analysis was used to evaluate the performance of circRNAs as diagnostic biomarkers for pSS. RESULTS: Circ-IQGAP2 and circ-ZC3H6 had significantly upregulated expression in the MSGs of pSS patients, and this elevated expression was confirmed by quantitative real-time PCR of plasma exosome RNA. The expression of these circRNAs also showed significant correlation with both clinical features, serum IgG level and MSG focus scores. Receiver operating characteristic analysis showed that the indices comprised of both the two circRNAs and clinical features were better able to distinguish pSS from non-pSS subjects with high mean areas under the curve of 0.93 in the MSGs and 0.92 in the plasma exosomes. CONCLUSION: This study indicated the potential roles of circ-IQGAP2 and circ-ZC3H6 as noninvasive biomarkers for the diagnosis of pSS.

Draft Genome Sequence of a Potential Organic Phosphorus-Degrading Bacterium Brevibacterium frigoritolerans GD44, Isolated from Radioactive Soil in Xinjiang, China.

Brevibacterium frigoritolerans, a strain quite potential use in environmental pollution, is also able to degrade the pesticide phorate. Here, we report a strain isolated from radioactive soil in the Xinjiang Uygur Autonomous Region of China. The genome of strain GD44 encompasses 5,471,331 base pairs with a GC content of 40.42%. The sequence was assembled into 1985 open reading frames (ORFs) encoding 5053 proteins. Sequence analysis identified the genes encoding enzymes related to the degradation of organophosphorus compounds such as esterase, phosphotransferase, C-P lyase, and alkaline phosphatase. The nitrate reductase gene was also found in GD44, which was associated with biosynthesis of silver nanoparticles used for bacteriostat. In addition, Antibiotic Resistance Ontology (ARO) genes accounted for 10.6%, including the vancomycin resistance gene cluster. Therefore, the whole-genome sequence of B. frigoritolerans GD44 will be beneficial for identifying and analyzing genes utilized for soil remediation and antibacterial agent, which will provide genetic evaluation for potential application in the future.

Complete Genome Sequence of Janibacter melonis M714, a Janus-Faced Bacterium with Both Human Health Impact and Industrial Applications.

Janibacter, a member of the Intrasporangiaceae family of Actinobacteria, is a Janus-faced bacterium that has both antibiotic resistance/pathogenicity and the ability to degrade pollutants, with significant research value. Here, we isolated the novel strain Janibacter melonis M714 from an irradiated area in Xinjiang Uygur Autonomous Region, China. J. melonis M714 contains one circular chromosome of 3,426,637 bp with a GC content of 72.98% and one plasmid of 54,436 bp with a GC content of 67.80%. The genome of J. melonis M714 contains 2,859 CDSs, 47 tRNA genes, and 6 rRNA genes. Genome assembly and annotation indicated that strain M714 has a high GC content and contains multiple notable functional genes, including a beta-lactam resistance gene and dioxygenase gene, which may be the key determinants of the strain's antibiotic resistance and xenobiotic degradation ability, respectively. The whole genome sequences of J. melonis M714 provide information that is useful for its potential applications in the degradation of pollutants and environmental remediation.

One-Step Synthesis of Monodispersed Mesoporous Carbon Nanospheres for High-Performance Flexible Quasi-Solid-State Micro-Supercapacitors.

Cost-effective synthesis of carbon nanospheres with a desirable mesoporous network for diversified energy storage applications remains a challenge. Herein, a direct templating strategy is developed to fabricate monodispersed N-doped mesoporous carbon nanospheres (NMCSs) with an average particle size of 100 nm, a pore diameter of 4 nm, and a specific area of 1093 m2 g-1 . Hexadecyl trimethyl ammonium bromide and tetraethyl orthosilicate not only play key roles in the evolution of mesopores but also guide the assembly of phenolic resins to generate carbon nanospheres. Benefiting from the high surface area and optimum mesopore structure, NMCSs deliver a large specific capacitance up to 433 F g-1 in 1 m H2 SO4 . The NMCS electrodes-based symmetric sandwich supercapacitor has an output voltage of 1.4 V in polyvinyl alcohol/H2 SO4 gel electrolyte and delivers an energy density of 10.9 Wh kg-1 at a power density of 14014.5 W kg-1 . Notably, NMCSs can be directly applied through the mask-assisted casting technique by a doctor blade to fabricate micro-supercapacitors. The micro-supercapacitors exhibit excellent mechanical flexibility, long-term stability, and reliable power output.

The effects of neuromedin S on the hypothalamic-pituitary-testicular axis in male pigs in vitro.

Evidence has shown that neuromedin S (NMS) and its receptor (NMU2R) are expressed in the hypothalamus, pituitary, and testis of pigs. To determine the potential mechanisms of NMS, we systematically investigated the direct effects of NMS on the hypothalamic-pituitary-testicular (HPT) axis of male pigs in vitro. We initially confirmed that NMU2R distributed in isolated hypothalamic cells, anterior pituitary cells and Leydig cells using immunocytochemistry. Subsequently we investigated the direct effects of NMS on hormone secretion from cells (anterior pituitary cells and Leydig cells) treated with different doses of NMS. The results showed that NMS increase the release of LH and FSH from anterior pituitary cells and testosterone from Leydig cells. NMS up-regulated the expression of NMU2R and GnRH mRNAs in hypothalamic cells, NMU2R, LH and FSH mRNAs in anterior pituitary cells, and NMU2R, STAR, P450 and 3ß-HSD mRNAs and the expression of PCNA and Cyclin B1 protein in Leydig cells; moreover, it down-regulated the expression of GnIH mRNA in hypothalamic cells. Using immunofluorescence staining and confocal microscopy, we also demonstrated the colocalization of NMU2R and AR or GnIH in Leydig cells. These data in vitro indicated that NMS may regulate the release and/or synthesis of LH, FSH and testosterone at different levels of the reproductive axis through NMU2R, which provided novel evidence of the potential roles of NMS in regulation of pig reproduction.

Sortase A-aided Escherichia coli expression system for functional osteoprotegerin cysteine-rich domain.

As a natural inhibitor of the receptor activator of nuclear factor-кB ligand (RANKL), osteprotegerin (OPG) is considered a promising treatment for metabolic bone diseases. Typical approaches for preparing recombinant OPG or its derivatives employ eukaryotic expression systems. Due to the advantages of a prokaryotic expression system, which include its convenience, low cost, and abundant production, in this study, we establish a strategy for preparing functional OPG using the Escherichia coli expression system. After initial failures in preparation of OPG and its truncation, OPG cysteine-rich domain (OPG-CRD/OPGT) by using pET and pGEX vectors, we constructed a sortase A (SrtA)-aided E. coli expression system, in which the expressed protein was a self-cleaving SrtA fusion protein. Using this system, we successfully prepared the recombinant OPGT protein. The BIAcore analyses indicated that the prepared OPGT had high affinities in binding with RANKL and TRAIL. Cell experiments confirmed the inhibitory effects of the prepared OPGT on RANKL-induced osteoclast differentiation and TRAIL-induced tumor cell apoptosis. The sortase A-aided E. coli expression system for OPGT established in this study may contribute to further studies and commercial applications of OPG.

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 ß-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.