The Experts below are selected from a list of 14556 Experts worldwide ranked by ideXlab platform
Aaron J. W. Hsueh - One of the best experts on this subject based on the ideXlab platform.
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characterization of novel splice variants of lgr7 and lgr8 reveals that receptor signaling is mediated by their unique low density lipoprotein class a modules
Journal of Biological Chemistry, 2006Co-Authors: Daniel Scott, Aaron J. W. Hsueh, Sharon Layfield, Satoko Sudo, Yan Yan, Geoffrey W Tregear, Ross A D BathgateAbstract:The relaxin and insulin-like peptide 3 receptors, LGR7 and LGR8, respectively, are unique members of the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family, because they possess an N-terminal motif with homology to the low density lipoprotein class A (LDLa) modules. By characterizing several LGR7 and LGR8 splice variants, we have revealed that the LDLa module directs ligand-activated cAMP signaling. The LGR8-short variant encodes an LGR8 receptor lacking the LDLa module, whereas LGR7-truncate, LGR7-truncate-2, and LGR7-truncate-3 all encode truncated secreted proteins retaining the LGR7 LDLa module. LGR8-short and an engineered LGR7 variant missing its LDLa module, LGR7-short, bound to their respective ligands with high affinity but lost their ability to signal via stimulation of intracellular cAMP accumulation. Conversely, secreted LGR7-truncate protein with the LDLa module was able to block relaxin-induced LGR7 cAMP signaling and did so without compromising the ability of LGR7 to bind to relaxin or be expressed on the cell membrane. Although the LDLa module of LGR7 was N-glycosylated at position Asn-14, an LGR7 N14Q mutant retained relaxin binding affinity and cAMP signaling, implying that glycosylation is not essential for optimal LDLa function. Using real-time PCR, the expression of mouse LGR7-truncate was detected to be high in, and specific to, the uterus of pregnant mice. The differential expression and evolutionary conservation of LGR7-truncate further suggests that it may also play an important role in vivo. This study highlights the essential role of the LDLa module in LGR7 and LGR8 function and introduces a novel model of GPCR regulation.
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leucine rich repeat containing g protein coupled receptor 4 null mice exhibit intrauterine growth retardation associated with embryonic and perinatal lethality
Molecular Endocrinology, 2004Co-Authors: Sabine Mazerbourg, Satoko Sudo, Donna M Bouley, Cynthia Klein, Jian Zhang, Kazuhiro Kawamura, Lisa V Goodrich, Helen Rayburn, Marc Tessierlavigne, Aaron J. W. HsuehAbstract: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 β-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 immunohistoch...
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h3 relaxin is a specific ligand for lgr7 and activates the receptor by interacting with both the ectodomain and the exoloop 2
Journal of Biological Chemistry, 2003Co-Authors: Satoko Sudo, Sharon Layfield, Tania Ferraro, Shinya Nishi, Jin Kumagai, Ross A D Bathgate, Aaron J. W. HsuehAbstract:Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) represent a unique subgroup of G protein-coupled receptors with a large ectodomain. Recent studies demonstrated that relaxin activates two orphan LGRs, LGR7 and LGR8, whereas INSL3/Leydig insulin-like peptide specifically activates LGR8. Human relaxin 3 (H3 relaxin) was recently discovered as a novel ligand for relaxin receptors. Here, we demonstrate that H3 relaxin activates LGR7 but not LGR8. Taking advantage of the overlapping specificity of these three ligands for the two related LGRs, chimeric receptors were generated to elucidate the mechanism of ligand activation of LGR7. Chimeric receptor LGR7/8 with the ectodomain from LGR7 but the transmembrane region from LGR8 maintains responsiveness to relaxin but was less responsive to H3 relaxin based on ligand stimulation of cAMP production. The decreased ligand signaling was accompanied by decreases in the ability of H3 relaxin to compete for (33)P-relaxin binding to the chimeric receptor. However, replacement of the exoloop 2, but not exoloop 1 or 3, of LGR7 to the chimeric LGR7/8 restored ligand binding and receptor-mediated cAMP production. These results suggested that activation of LGR7 by H3 relaxin involves specific binding of the ligand to both the ectodomain and the exoloop 2, thus providing a model with which to understand the molecular basis of ligand signaling for this unique subgroup of G protein-coupled receptors.
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insl3 leydig insulin like peptide activates the lgr8 receptor important in testis descent
Journal of Biological Chemistry, 2002Co-Authors: Jin Kumagai, Sheau Yu Hsu, Ross A D Bathgate, Hirotaka Matsumi, Jaesook Roh, John D Wade, Aaron J. W. HsuehAbstract:Several orphan G protein-coupled receptors homologous to gonadotropin and thyrotropin receptors have recently been identified and named as LGR4-8. INSL3, also known as Leydig insulin-like peptide or relaxin-like factor, is a relaxin family member expressed in testis Leydig cells and ovarian theca and luteal cells. Male mice mutant for INSL3 exhibit cryptorchidism or defects in testis descent due to abnormal gubernaculum development whereas overexpression of INSL3 induces ovary descent in transgenic females. Because transgenic mice missing the LGR8 gene are also cryptorchid, INSL3 was tested as the ligand for LGR8. Here, we show that treatment with INSL3 stimulated cAMP production in cells expressing recombinant LGR8 but not LGR7. In addition, interactions between INSL3 and LGR8 were demonstrated following ligand receptor cross-linking. Northern blot analysis indicated that the LGR8 transcripts are expressed in gubernaculum whereas treatment of cultured gubernacular cells with INSL3 stimulated cAMP production and thymidine incorporation. The present study identified the ligand for an orphan G protein-coupled receptor based on common phenotypes of ligand and receptor null mice. Demonstration of INSL3 as the ligand for LGR8 facilitates understanding of the mechanism of testis descent and allows studies on the role of INSL3 in gonadal and other physiological processes.
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the ectodomain of the luteinizing hormone receptor interacts with exoloop 2 to constrain the transmembrane region studies using chimeric human and fly receptors
Journal of Biological Chemistry, 2002Co-Authors: Shinya Nishi, Brian K. Kobilka, Koji Nakabayashi, Aaron J. W. HsuehAbstract:Lutropin (LH) and follitropin (FSH) receptors belong to a group of leucine-rich repeat-containing, G protein-coupled receptors (LGRs) found in vertebrates and flies. We fused the ectodomain of human LH or FSH receptors to the transmembrane region of fly LGR2. The chimeric human/fly receptors, unlike their wild type counterparts, exhibited ligand-independent constitutive activity. Because ectodomains likely interact with exoloops to constrain the receptors, individual exoloops of the chimeric receptor containing the ectodomain of the LH receptor and transmembrane region of fly LGR2 was replaced with LH receptor sequences. Chimeric receptors with the ectodomain and exoloop 2, but not exoloop 1 or 3, from LH receptors showed decreases in constitutive activity, but ligand treatment stimulated cAMP production. Furthermore, substitution of key resides in the hinge region of fly LGR2 with LH receptor sequences led to constitutive receptor activation; however, concomitant substitution of the homologous exoloop 2 of the LH receptor decreased G(s) coupling. These results suggest that the hinge region of the LH receptor interacts with exoloop 2 to constrain the receptor in an inactive conformation whereas ligand binding relieves this constraint, leading to G(s) activation.
Nick Barker - One of the best experts on this subject based on the ideXlab platform.
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LGR5 marks stem progenitor cells in ovary and tubal epithelia
Nature Cell Biology, 2014Co-Authors: Shawna Tan, Marc Leushacke, Yada Swathi, Gurmit Singh, Pamela Rizk, Tuan Zea Tan, Ruby Yunju Huang, Nick BarkerAbstract:The ovary surface epithelium (OSE) undergoes ovulatory tear and remodelling throughout life. Resident stem cells drive such tissue homeostasis in many adult epithelia, but their existence in the ovary has not been definitively proven. LGR5 marks stem cells in multiple epithelia. Here we use reporter mice and single-molecule fluorescent in situ hybridization to document candidate LGR5(+) stem cells in the mouse ovary and associated structures. LGR5 is broadly expressed during ovary organogenesis, but becomes limited to the OSE in neonate life. In adults, LGR5 expression is predominantly restricted to proliferative regions of the OSE and mesovarian-fimbria junctional epithelia. Using in vivo lineage tracing, we identify embryonic and neonate LGR5(+) populations as stem/progenitor cells contributing to the development of the OSE cell lineage, as well as epithelia of the mesovarian ligament and oviduct/fimbria. Adult LGR5(+) populations maintain OSE homeostasis and ovulatory regenerative repair in vivo. Thus, LGR5 marks stem/progenitor cells of the ovary and tubal epithelia.
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LGR5 positivity defines stem like cells in colorectal cancer
Carcinogenesis, 2014Co-Authors: Daniela Hirsch, Nick Barker, Hans Clevers, Nicole Mcneil, Jordi Camps, Katherine Mckinnon, Thomas Ried, Timo GaiserAbstract:Like normal colorectal epithelium, colorectal carcinomas (CRCs) are organized hierarchically and include populations of cells with stem-like properties. Leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) is associated with these stem cells in normal colorectal epithelium; however, the precise function of LGR5 in CRC remains largely unknown. Here, we analyzed the functional and molecular consequences of short hairpin RNA-mediated silencing of LGR5 in CRC cell lines SW480 and HT-29. Additionally, we exposed LGR5-EGFP-IRES-CreERT2 mice to azoxymethane/dextrane sodium sulfate (AOM/DSS), which induces inflammation-driven colon tumors. Tumors were then flow-sorted into fractions of epithelial cells that expressed high or low levels of LGR5 and were molecularly characterized using gene expression profiling and array comparative genomic hybridization. Silencing of LGR5 in SW480 CRC cells resulted in a depletion of spheres but did not affect adherently growing cells. Spheres expressed higher levels of several stem cell-associated genes than adherent cells, including LGR5. Silencing of LGR5 reduced proliferation, migration and colony formation in vitro and tumorigenicity in vivo. In accordance with these results, NOTCH signaling was downregulated upon LGR5 silencing. In AOM/DSS-induced colon tumors, LGR5 high cells showed higher levels of several stem cell-associated genes and higher Wnt signaling than LGR5 low tumor cells and LGR5 high normal colon cells. Array comparative genomic hybridization revealed no genomic imbalances in either tumor cell fraction. Our data elucidate mechanisms that define the role of LGR5 as a marker for stem-like cells in CRC.
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spasmolytic polypeptide expressing metaplasia spem in the gastric oxyntic mucosa does not arise from LGR5 expressing cells
Gut, 2012Co-Authors: Ki Taek Nam, Nick Barker, Ryan L Oneal, Robert J Coffey, Paul E Finke, James R GoldenringAbstract:Objective Metaplastic lineages in the oxyntic mucosa of the stomach are critical preneoplastic precursors of gastric cancer. Recent studies have demonstrated that spasmolytic polypeptide-expressing metaplasia (SPEM) in the mouse oxyntic mucosa arises from transdifferentiation of mature gastric chief cells. Other investigations of intestinal progenitor cells have shown that cells demonstrating transcriptional activity for leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5) in the intestine, colon and gastric antrum function as adult stem cells. We have now investigated whether cells demonstrating LGR5 transcriptional activity in the oxyntic mucosa of mice might be responsible for development of metaplasia. Design LGR5-EGFP-IRES-Cre ERT2/+ ;Rosa26R mice were used to examine the distribution of LGR5 transcriptionally active cells in the normal oxyntic mucosa as well as after treatment with DMP-777 or L-635 to induce acute SPEM. Lineage mapping was performed to determine if LGR5-expressing cells gave rise to SPEM. Results Cells expressing transcriptional activity for LGR5 in the oxyntic mucosa were present as scattered rare cells only along the lesser curvature of the stomach. These cells also stained for markers of chief cells (intrinsic factor and pepsinogen) but never showed any staining for proliferative markers (Ki-67). In LGR5-EGFP-IRES-Cre ERT2/+ ;Rosa26R mice induced with tamoxifen, treatment with either DMP-777 or L-635 to induce acute oxyntic atrophy caused induction of SPEM, but no lineage mapping into SPEM from LGR5-expressing cells was observed. Conclusion The results indicate that, while chief cells with LGR5 transcriptional activity are present along the lesser curvature of the gastric oxyntic mucosa, they are not responsible for production of metaplasia.
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developmental stage specific contribution of LGR5 cells to basal and luminal epithelial lineages in the postnatal mammary gland
The Journal of Pathology, 2012Co-Authors: Karin E De Visser, Nick Barker, Hans Clevers, Metamia Ciampricotti, Ewa M Michalak, David Weimin Tan, Ewoud N Speksnijder, Cheeising Hau, Jos JonkersAbstract:The leucine-rich repeat-containing heterotrimeric guanine nucleotide-binding protein-coupled receptor 5 (LGR5) has been identified as a marker of cycling stem cells in several epithelial tissues, including small intestine, colon, stomach and hair follicle. To investigate whether LGR5 also marks mammary epithelial stem cells, we performed in situ lineage-tracing studies and mammary gland reconstitutions with LGR5-expressing mammary epithelial cells. Interestingly, the LGR5 progeny population in mammary epithelium switches from the luminal to the myoepithelial compartment during the first 12 days of postnatal development, likely reflecting local changes in Wnt signalling. Together, our findings point to a stage-specific contribution of LGR5-expressing cells to luminal and basal epithelial lineages during postnatal mammary gland development.
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LGR5 ve stem progenitor cells contribute to nephron formation during kidney development
Cell Reports, 2012Co-Authors: Nick Barker, Pekka Kujala, Meritxell Huch, Marc Van De Wetering, Maarten B Rookmaaker, Marc Leushacke, Hugo J Snippert, Shawna Tan, Johan H Van Es, Richard PoulsomAbstract:Multipotent stem cells and their lineage-restricted progeny drive nephron formation within the developing kidney. Here, we document expression of the adult stem cell marker LGR5 in the developing kidney and assess the stem/progenitor identity of LGR5(+ve) cells via in vivo lineage tracing. The appearance and localization of LGR5(+ve) cells coincided with that of the S-shaped body around embryonic day 14. LGR5 expression remained restricted to cell clusters within developing nephrons in the cortex until postnatal day 7, when expression was permanently silenced. In vivo lineage tracing identified LGR5 as a marker of a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb of Henle's loop and distal convoluted tubule. The LGR5 surface marker and experimental models described here will be invaluable for deciphering the contribution of early nephron stem cells to developmental defects and for isolating human nephron progenitors as a prerequisite to evaluating their therapeutic potential.
Hans Clevers - One of the best experts on this subject based on the ideXlab platform.
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LGR5 positivity defines stem like cells in colorectal cancer
Carcinogenesis, 2014Co-Authors: Daniela Hirsch, Nick Barker, Hans Clevers, Nicole Mcneil, Jordi Camps, Katherine Mckinnon, Thomas Ried, Timo GaiserAbstract:Like normal colorectal epithelium, colorectal carcinomas (CRCs) are organized hierarchically and include populations of cells with stem-like properties. Leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) is associated with these stem cells in normal colorectal epithelium; however, the precise function of LGR5 in CRC remains largely unknown. Here, we analyzed the functional and molecular consequences of short hairpin RNA-mediated silencing of LGR5 in CRC cell lines SW480 and HT-29. Additionally, we exposed LGR5-EGFP-IRES-CreERT2 mice to azoxymethane/dextrane sodium sulfate (AOM/DSS), which induces inflammation-driven colon tumors. Tumors were then flow-sorted into fractions of epithelial cells that expressed high or low levels of LGR5 and were molecularly characterized using gene expression profiling and array comparative genomic hybridization. Silencing of LGR5 in SW480 CRC cells resulted in a depletion of spheres but did not affect adherently growing cells. Spheres expressed higher levels of several stem cell-associated genes than adherent cells, including LGR5. Silencing of LGR5 reduced proliferation, migration and colony formation in vitro and tumorigenicity in vivo. In accordance with these results, NOTCH signaling was downregulated upon LGR5 silencing. In AOM/DSS-induced colon tumors, LGR5 high cells showed higher levels of several stem cell-associated genes and higher Wnt signaling than LGR5 low tumor cells and LGR5 high normal colon cells. Array comparative genomic hybridization revealed no genomic imbalances in either tumor cell fraction. Our data elucidate mechanisms that define the role of LGR5 as a marker for stem-like cells in CRC.
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the r spondin LGR5 rnf43 module regulator of wnt signal strength
Genes & Development, 2014Co-Authors: Wim De Lau, Weng Chuan Peng, Piet Gros, Hans CleversAbstract:LGR5 was originally discovered as a common Wnt target gene in adult intestinal crypts and colon cancer. It was subsequently identified as an exquisite marker of multiple Wnt-driven adult stem cell types. LGR5 and its homologs, Lgr4 and Lgr6, constitute the receptors for R-spondins, potent Wnt signal enhancers and stem cell growth factors. The LGR5/R-spondin complex acts by neutralizing Rnf43 and Znrf3, two transmembrane E3 ligases that remove Wnt receptors from the stem cell surface. Rnf43/Znrf3 are themselves encoded by Wnt target genes and constitute a negative Wnt feedback loop. Thus, adult stem cells are controlled by an intricate interplay of potent Wnt agonists, antagonists, and anti-antagonists.
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Structure of Stem Cell Growth Factor R-spondin 1 in Complex with the Ectodomain of Its Receptor LGR5
Cell Reports, 2013Co-Authors: Weng Chuan Peng, Federico Forneris, Meritxell Huch, Joke C. M. Granneman, Hans Clevers, Piet GrosAbstract:Summary Leucine-rich repeat-containing G protein-coupled receptors 4–6 (LGR4–LGR6) are receptors for R-spondins, potent Wnt agonists that exert profound trophic effects on Wnt-driven stem cells compartments. We present crystal structures of a signaling-competent fragment of R-spondin 1 (Rspo1) at a resolution of 2.0 A and its complex with the LGR5 ectodomain at a resolution of 3.2 A. Ecto-LGR5 binds Rspo1 at its concave leucine-rich-repeat (LRR) surface, forming a dimeric 2:2 complex. Fully conserved residues on LGR4–LGR6 explain promiscuous binding of R-spondins. A phenylalanine clamp formed by Rspo1 Phe106 and Phe110 pinches Ala190 of LGR5 and is critical for binding. Mutations related to congenital anonychia reduce signaling, but not binding of Rspo1 to LGR5. Furthermore, antibody binding to the extended loop of the C-terminal LRR cap of LGR5 activates signaling in a ligand-independent manner. Thus, our data reveal binding of R-spondins to conserved sites on LGR4–LGR6 and, in analogy to FSHR and related receptors, suggest a direct signaling role for LGR4–LGR6 in addition to its formation of Wnt receptor and coreceptor complexes.
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monoclonal antibodies against LGR5 identify human colorectal cancer stem cells
Stem Cells, 2012Co-Authors: Kristel Kemper, Wim De Lau, Hans Clevers, Pramudita R Prasetyanti, Hans M Rodermond, Jan Paul MedemaAbstract:In colorectal cancer (CRC), a subpopulation of tumor cells, called cancer stem cell (CSC) fraction, is suggested to be responsible for tumor initiation, growth, and metastasis. The search for a reliable marker to identify these CSCs is ongoing as current markers, like CD44 and CD133, are more broadly expressed and therefore are not highly selective and currently also lack function in CSC biology. Here, we analyzed whether the Wnt target LGR5, which has earlier been identified as a marker for murine intestinal stem cells, could potentially serve as a functional marker for CSCs. Fluorescence-activated cell sorting-based detection of LGR5, using three newly developed antibodies, on primary colorectal tumor cells revealed a clear subpopulation of Epcam+ LGR5+ cells. Similarly, primary CRC-derived spheroid cultures, known to be enriched for CSCs, contain high levels of LGR5+ cells, which decrease upon in vitro differentiation of these CSCs. Selection of the LGR5(high) CRC cells identified the clonogenic fraction in vitro as well as the tumorigenic population in vivo. Finally, we confirm that LGR5 expression is dependent on the Wnt pathway and show that LGR5 overexpression induces clonogenic growth. We thus provide evidence that LGR5 is, next to a functional intestinal stem cell marker, a selective marker for human colorectal CSCs.
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developmental stage specific contribution of LGR5 cells to basal and luminal epithelial lineages in the postnatal mammary gland
The Journal of Pathology, 2012Co-Authors: Karin E De Visser, Nick Barker, Hans Clevers, Metamia Ciampricotti, Ewa M Michalak, David Weimin Tan, Ewoud N Speksnijder, Cheeising Hau, Jos JonkersAbstract:The leucine-rich repeat-containing heterotrimeric guanine nucleotide-binding protein-coupled receptor 5 (LGR5) has been identified as a marker of cycling stem cells in several epithelial tissues, including small intestine, colon, stomach and hair follicle. To investigate whether LGR5 also marks mammary epithelial stem cells, we performed in situ lineage-tracing studies and mammary gland reconstitutions with LGR5-expressing mammary epithelial cells. Interestingly, the LGR5 progeny population in mammary epithelium switches from the luminal to the myoepithelial compartment during the first 12 days of postnatal development, likely reflecting local changes in Wnt signalling. Together, our findings point to a stage-specific contribution of LGR5-expressing cells to luminal and basal epithelial lineages during postnatal mammary gland development.
Kendra S Carmon - One of the best experts on this subject based on the ideXlab platform.
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Drug Conjugates of Antagonistic R‑Spondin 4 Mutant for Simultaneous Targeting of Leucine-Rich Repeat-Containing G Protein-Coupled Receptors 4/5/6 for Cancer Treatment
'American Chemical Society (ACS)', 2021Co-Authors: Jie Cui, Yukimatsu Toh, Soohyun Park, Joan Jacob, Sheng Pan, Kendra S CarmonAbstract:LGR4–6 (leucine-rich repeat-containing G-protein-coupled receptors 4, 5, and 6) are three related receptors with an upregulated expression in gastrointestinal cancers to various extents, and LGR5 is enriched in cancer stem cells. Antibody–drug conjugates (ADCs) targeting LGR5 showed a robust antitumor effect in vivo but could not eradicate tumors due to plasticity of LGR5-positive cancer cells. As LGR5-negative cancer cells often express LGR4 or LGR6 or both, we reasoned that simultaneous targeting of all three LGRs may provide a more effective approach. R-spondins (RSPOs) bind to LGR4–6 with high affinity and potentiate Wnt signaling. We identified an RSPO4 furin domain mutant (Q65R) that retains potent LGR binding but no longer potentiates Wnt signaling. Drug conjugates of a peptibody comprising the RSPO4 mutant and IgG1-Fc showed potent cytotoxic effects on cancer cell lines expressing any LGR in vitro and suppressed tumor growth in vivo without inducing intestinal enlargement or other adverse effects
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LGR5 receptor promotes cell cell adhesion in stem cells and colon cancer cells via the iqgap1 rac1 pathway
Journal of Biological Chemistry, 2017Co-Authors: Kendra S Carmon, Xing Gong, Anthony Thomas, Catherine M Moore, Ikuo Masuho, David J Timson, Kirill A Martemyanov, Qingyun LiuAbstract:Leucine-rich repeat-containing G protein–coupled receptor 5 (LGR5) is a bona fide marker of adult stem cells in several epithelial tissues, most notably in the intestinal crypts, and is highly up-regulated in many colorectal, hepatocellular, and ovarian cancers. LGR5 activation by R-spondin (RSPO) ligands potentiates Wnt/β-catenin signaling in vitro; however, deletion of LGR5 in stem cells has little or no effect on Wnt/β-catenin signaling or cell proliferation in vivo. Remarkably, modulation of LGR5 expression has a major impact on the actin cytoskeletal structure and cell adhesion in the absence of RSPO stimulation, but the molecular mechanism is unclear. Here, we show that LGR5 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1), an effector of Rac1/CDC42 GTPases, in the regulation of actin cytoskeleton dynamics and cell–cell adhesion. Specifically, LGR5 decreased levels of IQGAP1 phosphorylation at Ser-1441/1443, leading to increased binding of Rac1 to IQGAP1 and thus higher levels of cortical F-actin and enhanced cell–cell adhesion. LGR5 ablation in colon cancer cells and crypt stem cells resulted in loss of cortical F-actin, reduced cell–cell adhesion, and disrupted localization of adhesion-associated proteins. No evidence of LGR5 coupling to any of the four major subtypes of heterotrimeric G proteins was found. These findings suggest that LGR5 primarily functions via the IQGAP1–Rac1 pathway to strengthen cell–cell adhesion in normal adult crypt stem cells and colon cancer cells.
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LGR5 targeted antibody drug conjugate eradicates gastrointestinal tumors and prevents recurrence
Molecular Cancer Therapeutics, 2016Co-Authors: Xing Gong, Qingyun Liu, Ali Azhdarinia, Sukhen C Ghosh, Wei Xiong, Kendra S CarmonAbstract:Gastrointestinal cancer is one of the leading causes of cancer-related mortality in men and women worldwide. The adult stem cell marker LGR5 (leucine-rich repeat-containing, G protein-coupled receptor 5) is highly expressed in a significant fraction of gastrointestinal tumors of the colon, liver, pancreas, and stomach, relative to normal tissues. LGR5 is located on the cell surface and undergoes rapid, constitutive internalization independent of ligand. Furthermore, LGR5-high cancer cells have been shown to exhibit the properties of tumor-initiating cells or cancer stem cells (CSC). On the basis of these attributes, we generated two LGR5-targeting antibody-drug conjugates (ADC) by tethering the tubulin-inhibiting cytotoxic drug monomethyl auristatin E to a highly specific anti-LGR5 mAb via a protease cleavable or noncleavable chemical linker and compared them in receptor binding, cell internalization, and cytotoxic efficacy in cancer cells. Here, we show that both ADCs bind LGR5 with high specificity and equivalent nanomolar affinity and rapidly internalize to the lysosomes of LGR5-expressing gastrointestinal cancer cells. The anti-LGR5 ADCs effectively induced cytotoxicity in LGR5-high gastrointestinal cancer cells, but not in LGR5-negative or -knockdown cancer cell lines. Overall, we demonstrate that the cleavable ADC exhibited higher potency in vitro and was able to eradicate tumors and prevent recurrence in a xenograft model of colon cancer. These findings provide preclinical evidence for the potential of LGR5-targeting ADCs as effective new therapeutics for the treatment and eradication of gastrointestinal tumors and CSCs with high LGR5 expression. Mol Cancer Ther; 15(7); 1580-90. ©2016 AACR.
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LGR5 interacts and cointernalizes with wnt receptors to modulate wnt β catenin signaling
Molecular and Cellular Biology, 2012Co-Authors: Kendra S Carmon, Xing Gong, Anthony Thomas, Qiushi Lin, Qingyun LiuAbstract:LGR5, a seven-transmembrane domain receptor of the rhodopsin family, is a Wnt target gene and a bona fide marker of adult stem cells in the gastrointestinal tract and hair follicle bulge. Recently, we and others demonstrated that LGR5 and its homologues function as receptors of the R-spondin family of stem cell factors to potentiate Wnt/β-catenin signaling. However, the mechanism of how LGR5 enhances the signaling output remains unclear. Here we report that following costimulation with the ligands R-spondin1 and Wnt3a, LGR5 interacts and forms a supercomplex with the Wnt coreceptors LRP6 and Fzd5 which is rapidly internalized and then degraded. Internalization of LGR5 is mediated through a dynamin- and clathrin-dependent pathway. Inhibition of this endocytic process has no effect on LGR5 signaling. Deletion of the C-terminal tail of LGR5 maintains its ability to interact with LRP6, yet this LGR5 mutant exhibits increased signaling activity and a decreased rate of endocytosis in response to R-spondin1 compared to the wild-type receptor. This study provides direct evidence that LGR5 becomes part of the Wnt signaling complex at the membrane level to enhance Wnt/β-catenin signaling. However, internalization of LGR5 does not appear to be essential for potentiating the canonical Wnt signaling pathway.
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r spondins function as ligands of the orphan receptors lgr4 and LGR5 to regulate wnt β catenin signaling
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Kendra S Carmon, Xing Gong, Anthony ThomasAbstract:The Wnt/β-catenin signaling system plays essential roles in embryonic development and in the self-renewal and maintenance of adult stem cells. R-spondins (RSPOs) are a group of secreted proteins that enhance Wnt/β-catenin signaling and have pleiotropic functions in development and stem cell growth. LGR5, an orphan receptor of the G protein-coupled receptor (GPCR) superfamily, is specifically expressed in stem cells of the intestinal crypt and hair follicle. Knockout of LGR5 in the mouse results in neonatal lethality. LGR4, a receptor closely related to LGR5, also has essential roles in development, as its knockout leads to reduced viability and retarded growth. Overexpression of both receptors has been reported in several types of cancer. Here we demonstrate that LGR4 and LGR5 bind the R-spondins with high affinity and mediate the potentiation of Wnt/β-catenin signaling by enhancing Wnt-induced LRP6 phosphorylation. Interestingly, neither receptor is coupled to heterotrimeric G proteins or to β-arrestin when stimulated by the R-spondins, indicating a unique mechanism of action. The findings provide a basis for stem cell-specific effects of Wnt/β-catenin signaling and for the broad range of functions LGR4, LGR5, and the R-spondins have in normal and malignant growth.
Toshiro Sato - One of the best experts on this subject based on the ideXlab platform.
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visualization and targeting of LGR5 human colon cancer stem cells
Nature, 2017Co-Authors: Mariko Shimokawa, Yuki Ohta, Shingo Nishikori, Mami Matano, Ai Takano, Masayuki Fujii, Shoichi Date, Shinya Sugimoto, Takanori Kanai, Toshiro SatoAbstract:The cancer stem cell (CSC) theory highlights a self-renewing subpopulation of cancer cells that fuels tumour growth. The existence of human CSCs is mainly supported by xenotransplantation of prospectively isolated cells, but their clonal dynamics and plasticity remain unclear. Here, we show that human LGR5+ colorectal cancer cells serve as CSCs in growing cancer tissues. Lineage-tracing experiments with a tamoxifen-inducible Cre knock-in allele of LGR5 reveal the self-renewal and differentiation capacity of LGR5+ tumour cells. Selective ablation of LGR5+ CSCs in LGR5-iCaspase9 knock-in organoids leads to tumour regression, followed by tumour regrowth driven by re-emerging LGR5+ CSCs. KRT20 knock-in reporter marks differentiated cancer cells that constantly diminish in tumour tissues, while reverting to LGR5+ CSCs and contributing to tumour regrowth after LGR5+ CSC ablation. We also show that combined chemotherapy potentiates targeting of LGR5+ CSCs. These data provide insights into the plasticity of CSCs and their potential as a therapeutic target in human colorectal cancer.
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unlimited in vitro expansion of adult bi potent pancreas progenitors through the LGR5 r spondin axis
The EMBO Journal, 2013Co-Authors: Meritxell Huch, Marc Van De Wetering, Toshiro Sato, Sylvia F Boj, Paola Bonfanti, Cindy J M Loomans, Mozhdeh Sojoodi, Jurian Schuijers, Ana GracaninAbstract:LGR5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver LGR5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and LGR5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of LGR5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate LGR5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing LGR5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.
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in vitro expansion of single LGR5 liver stem cells induced by wnt driven regeneration
Nature, 2013Co-Authors: Meritxell Huch, Marc Van De Wetering, Johan H Van Es, Toshiro Sato, Craig Dorrell, Sylvia F Boj, Karien Hamer, Nobuo Sasaki, Milton J FinegoldAbstract:The Wnt target gene LGR5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single LGR5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that LGR5-lacZ is not expressed in healthy adult liver, however, small LGR5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new LGR5-IRES-creERT2 knock-in 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-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(-/-) mice. These findings indicate that previous observations concerning LGR5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.
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paneth cells constitute the niche for LGR5 stem cells in intestinal crypts
Nature, 2011Co-Authors: Toshiro Sato, Nick Barker, Marc Van De Wetering, Hugo J Snippert, Johan H Van Es, Noah F. Shroyer, Daniel E Stange, Robert G J Vries, Maaike Van Den Born, Hans CleversAbstract:Homeostasis of self-renewing small intestinal crypts results from neutral competition between LGR5 stem cells, which are small cycling cells located at crypt bottoms. LGR5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products such as lysozyme and cryptdins/defensins. Single LGR5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells. Here we find a close physical association of LGR5 stem cells with Paneth cells in mice, both in vivo and in vitro. CD24(+) Paneth cells express EGF, TGF-α, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing 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. We conclude that LGR5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.
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LGR5 ve stem cells drive self renewal in the stomach and build long lived gastric units in vitro
Cell Stem Cell, 2010Co-Authors: Nick Barker, Pekka Kujala, Meritxell Huch, Hugo J Snippert, Toshiro Sato, Daniel E Stange, Harry Begthel, Marc Van De Wetering, Maaike Van Den Born, Esther DanenbergAbstract:Summary The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene LGR5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated LGR5 expression in the stomach and assessed the stem cell potential of the LGR5 +ve cells by using in vivo lineage tracing. In neonatal stomach, LGR5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these LGR5 +ve cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single LGR5 +ve cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The LGR5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.