ArticleBiochemistry2013
Reconstitution of R-spondin:LGR4:ZNRF3 adult stem cell growth factor signaling complexes with recombinant proteins produced in Escherichia coli.
Article in Biochemistry, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 23 papers.
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Who cites it
23 citing papers in PubMed, 27 citations in OpenAlex.
- Regulation of RSPO3-LGR4 Signaling: Emerging Role in Inflammation Revealed by Network Analysis.Archives of clinical and biomedical research · 2025Article
- Regenerative human liver organoids (HLOs) in a pillar/perfusion plate for hepatotoxicity assays.bioRxiv : the preprint server for biology · 2024Article
- RSPO2 as Wnt signaling enabler: Important roles in cancer development and therapeutic opportunities.Genes & diseases · 2024Review
- Time- and cost-efficient bacterial expression and purification of potato apyrase.Protein expression and purification · 2023Article
- Arsenic exposure impairs intestinal stromal cells.Toxicology letters · 2022Article
- Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling.Nature communications · 2022Article
- Receptor control by membrane-tethered ubiquitin ligases in development and tissue homeostasis.Current topics in developmental biology · 2022Article
- The Role of LGR4 (GPR48) in Normal and Cancer Processes.International journal of molecular sciences · 2021Review
- R-Spondins 2 and 3 Are Overexpressed in a Subset of Human Colon and Breast Cancers.DNA and cell biology · 2021Article
- Unlike LGR4, LGR5 potentiates Wnt-β-catenin signaling without sequestering E3 ligases.Science signaling · 2020Article
- Molecular interaction of an antagonistic amylin analog with the extracellular domain of receptor activity-modifying protein 2 assessed by fluorescence polarization.Biophysical chemistry · 2020Article
- Production, purification and characterization of recombinant human R-spondin1 (RSPO1) protein stably expressed in human HEK293 cells.BMC biotechnology · 2020Article
- Organoid culture media formulated with growth factors of defined cellular activity.Scientific reports · 2019Article
- Tales from the crypt: intestinal niche signals in tissue renewal, plasticity and cancer.Open biology · 2018Review
- Differential activities and mechanisms of the four R-spondins in potentiating Wnt/β-catenin signaling.The Journal of biological chemistry · 2018Article
- Article
- N-Glycosylation of Human R-Spondin 1 Is Required for Efficient Secretion and Stability but Not for Its Heparin Binding Ability.International journal of molecular sciences · 2016Article
- Control of Wnt Receptor Turnover by R-spondin-ZNRF3/RNF43 Signaling Module and Its Dysregulation in Cancer.Cancers · 2016Review
- Crystal structure of R-spondin 2 in complex with the ectodomains of its receptors LGR5 and ZNRF3.Journal of structural biology · 2015Article
- The lymph node as a new site for kidney organogenesis.Stem cells translational medicine · 2015Article
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
Abstract
R-Spondins are secreted glycoproteins (RSPO1-RSPO4) that have proliferative effects on adult stem cells by potentiating Wnt signaling. RSPO actions are mediated by the leucine-rich repeat (LRR)-containing seven-transmembrane receptors LGR4-LGR6 and the transmembrane E3 ubiquitin ligases ZNRF3 and RNF43. Here, we present a methodology for the bacterial expression and purification of the signaling competent, cysteine-rich Fu1-Fu2 domains of the four human RSPOs, a fragment of the human LGR4 extracellular domain (ECD) containing LRR1-14, and the human ZNRF3 ECD. In a cell-based signaling assay, the nonglycosylated RSPOs enhanced low-dose Wnt3a signaling with potencies comparable to those of mammalian cell-produced RSPOs and RSPO2 and -3 were more potent than RSPO1 and -4. LGR4 LRR1-14 and ZNRF3 ECD inhibited RSPO2-enhanced Wnt3a signaling. The RSPOs bound LGR4 LRR1-14 with nanomolar affinities that decreased in the following order in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay: RSPO4 > RSPO2 > RSPO3 > RSPO1. RSPO-receptor interactions were further characterized with a native gel electrophoretic mobility shift assay, which corroborated the RSPO-LGR4 TR-FRET results and indicated that RSPOs weakly bound ZNRF3 with affinities that decreased in the following order: RSPO2 > RSPO3 > RSPO1. RSPO4:ZNRF3 complexes were not detected. Lastly, ternary RSPO:LGR4:ZNRF3 complexes were detected for RSPO2 and -3. Our results indicate that RSPO and LGR4 N-glycans are dispensable for function, demonstrate RSPO-mediated ternary complex formation, and suggest that the stronger signaling potencies of RSPO2 and -3 result from their strong binding of both receptors. Our unique protein production methodology may provide a cost-effective source of recombinant RSPOs for regenerative medicine applications.
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