Recombinant Human R-Spondin 3 Protein

Recombinant human R-Spondin 3 (Catalog # 3500-RS) has a molecular weight (MW) of 36.1 kDa as analyzed by SEC-MALS, suggesting that this protein is a monomer. MW may differ from predicted MW due to post-translational modifications (PTMs) present (i.e. Glycosylation).

Recombinant Human R-Spondin 3 (Catalog # 3500-RS), in the presence of Recombinant Mouse Wnt 3a (Catalog # 1324-WN; 5 ng/mL), induces activation of beta-catenin in HEK293T cells measured using the Topflash assay. The ED50 for this effect is 0.50-2.00 ng/mL.

The lot-to-lot consistency of Recombinant Human R-Spondin 3 (Catalog # 3500-RS) was assessed by testing the ability of three independent lots of the protein to stimulate activation of beta-Catenin using a TOPflash beta-Catenin/TCF reporter assay in the HEK293T human kidney cell line, in the presence of 5 ng/mL Recombinant Mouse Wnt-3a (1324-WN). Each trace on the graph represents data obtained from Recombinant Human R-Spondin 3 from a different manufacturing run. The ED50 for this effect is 0.500-2.00 ng/mL in the presence of 5 ng/mL Recombinant Mouse Wnt-3a.

• Reactivity: Hu
• Applications: Bioactivity

Recombinant Human R-Spondin 3 Protein Summary

• Additional Information: Analyzed by SEC-MALS
• Details of Functionality: Measured by its ability to induce Topflash reporter activity in HEK293T human embryonic kidney cells. The ED₅₀ for this effect is 0.500‑2.00 ng/mL in the presence of 5 ng/mL Recombinant Mouse Wnt‑3a (Catalog # 1324-WN).
• Source: Chinese Hamster Ovary cell line, CHO-derived human R-Spondin 3 protein
  Gln22-His272
• Accession #: Q9BXY4
• N-terminal Sequence: No results obtained: Gln22 predicted
• Protein/Peptide Type: Recombinant Proteins
• Gene: RSPO3
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Bioactivity
• Theoretical MW: 28.3 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 36-45 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with BSA as a carrier protein.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Reconstitution Instructions: Reconstitute at 100 μg/mL in PBS containing at least 0.1% of human or bovine serun albumin.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human R-Spondin 3 Protein

• Cristin 1
• CRISTIN1
• FLJ14440
• hPWTSR
• hRspo3
• Protein with TSP type-1 repeat
• PWTSR
• Roof plate-specific spondin-3
• RSPO3
• R-spondin 3 homolog (Xenopus laevis)
• RSpondin 3
• R-Spondin 3
• R-spondin-3
• Thrombospondin type-1 domain-containing protein 2
• thrombospondin, type I, domain containing 2
• THSD2

Background

R-Spondin 3 (RSPO3, roof plate-specific spondin 3), also called cysteine-rich and single thrombospondin domain containing-1 (Cristin 1), is an ~31 kDa secreted protein that shares ~40% amino acid (aa) identity with the other three R-Spondin family members (1, 2). All are positive modulators of Wnt/ beta -catenin signaling, but each has a distinct expression pattern (1-4). Like other R-spondins,R-Spondin 3 contains two adjacent cysteine-rich furin-like domains (aa 35-135) with one potential N-glycosylation site (aa 36), followed by a thrombospondin (TSP-1) motif (aa 147-207) and a region rich in basic residues (aa 211-269). Only the furin-like domains are needed for beta -catenin stabilization (2). Within aa 21-209, human R-Spondin 3 shares 93%, 92%, 97%, 96% and 92% aa identity with mouse, rat, equine, bovine and canine R-Spondin 3, respectively. Potential isoforms of 279 and 297 aa diverge at aa 210 and 276, respectively (5). Mouse R-Spondin 3 is critical for development of the placental labyrinthine layer, probably by promoting VEGF expression and thus vascular development (6, 7). It is also essential for expression of the placenta-specific transcription factor, Gcm1. In the mouse embryo, R-Spondin 3 is often expressed by or located near endothelial cells (6). It is found in the roof plate, tail, somites, otic vesicles, cephalic mesoderm, truncus arteriosus, atrioventricular canal of the developing heart, and strongly but transiently in developing limbs (4, 7). R-Spondins regulate Wnt/ beta -catenin by competing with the Wnt antagonist DKK-1 for binding to the Wnt co-receptors LRP-6 and Kremen, reducing their DKK-1-mediated internalization (8, 9). Reports differ on whether R-Spondins bind LRP-6 directly (8-10). R-Spondin 3 has also been identified as an oncogene (11).

1. Chen, J-Z. et al. (2002) Mol. Biol. Rep. 29:287.
2. Kim, K.-A. et al. (2008) Mol. Biol. Cell 19:2588.
3. Hendrickx, M. and L. Leyns (2008) Develop. Growth Differ. 50:229.
4. Nam, J.-S. et al. (2007) Gene Expr. Patterns 7:306.
5. Entrez Accession # EAW48114 and EAW48116.
6. Kazanskaya, O. et al. (2008) Development 135:3655.
7. Aoki, M. et al. (2007) Dev. Biol. 301:218.
8. Binnerts, M.E. et al. (2007) Proc. Natl. Acad. Sci. USA 104:14700.
9. Nam, J.-S. et al. (2006) J. Biol. Chem. 281:13247.
10. Wei, Q. et al. (2007) J. Biol. Chem. 282:15903.
11. Theodorou, V. et al. (2007) Nat. Genet. 6:759.

Publications for R-Spondin 3 (3500-RS)(18)

Publications using 3500-RS

• Song, X;Zhou, Z;Elmezayen, A;Wu, R;Yu, C;Gao, B;Minna, JD;Westover, KD;Zeh, HJ;Kroemer, G;Heasley, LE;Kang, R;Tang, D; SRC kinase drives multidrug resistance induced by KRAS-G12C inhibition Science advances 2024-12-13 [PMID: 39661665] (Bioassay, Human)
• K Nagano, K Yamana, H Saito, R Kiviranta, AC Pedroni, D Raval, C Niehrs, F Gori, R Baron R-spondin 3 deletion induces Erk phosphorylation to enhance Wnt signaling and promote bone formation in the appendicular skeleton Elife, 2022-11-02;11(0):. 2022-11-02 [PMID: 36321691] (Bioassay, Mouse)
• G Pizzolato, L Moparthi, S Söderholm, C Cantù, S Koch The oncogenic transcription factor FOXQ1 is a differential regulator of Wnt target genes Journal of Cell Science, 2022-10-10;0(0):. 2022-10-10 [PMID: 36124643] (Cell Culture, Human)
• JF Dekkers, EJ van Vliet, N Sachs, JM Rosenbluth, O Kopper, HG Rebel, EJ Wehrens, C Piani, JE Visvader, CS Verissimo, SF Boj, JS Brugge, H Clevers, AC Rios Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids Nature Protocols, 2021-03-10;0(0):. 2021-03-10 [PMID: 33692550] (Bioassay, Human)
• C Beaurivage, A Kanapeckai, C Loomans, KS Erdmann, J Stallen, RAJ Janssen Development of a human primary gut-on-a-chip to model inflammatory processes Scientific Reports, 2020-12-08;10(1):21475. 2020-12-08 [PMID: 33293676] (Bioassay, Human)
• NY Loh, JEN Minchin, KE Pinnick, M Verma, M Todor?evi?, N Denton, JE Moustafa, JP Kemp, CL Gregson, DM Evans, MJ Neville, KS Small, MI McCarthy, A Mahajan, JF Rawls, F Karpe, C Christodou RSPO3 impacts body fat distribution and regulates adipose cell biology in vitro Nat Commun, 2020-06-03;11(1):2797. 2020-06-03 [PMID: 32493999] (Bioassay, Human)
• X Li, B Pan, X Song, N Li, D Zhao, M Li, Z Zhao Breast cancer organoids from a patient with giant papillary carcinoma as a high-fidelity model Cancer Cell Int., 2020-03-18;20(0):86. 2020-03-18 [PMID: 32206037] (Bioassay, Human)
• M Zhang, M Haughey, NY Wang, K Blease, AM Kapoun, S Couto, I Belka, T Hoey, M Groza, J Hartke, B Bennett, J Cain, A Gurney, B Benish, P Castiglion, C Drew, J Lachowicz, L Carayannop, SD Nathan, J Distler, DA Brenner, K Hariharan, H Cho, W Xie Targeting the Wnt signaling pathway through R-spondin 3 identifies an anti-fibrosis treatment strategy for multiple organs PLoS ONE, 2020-03-11;15(3):e0229445. 2020-03-11 [PMID: 32160239] (IHC Control, Human)
• PG Boone, LK Rochelle, JD Ginzel, V Lubkov, WL Roberts, PJ Nicholls, C Bock, ML Flowers, RJ von Furste, BR Stripp, P Agarwal, AD Borowsky, RD Cardiff, LS Barak, MG Caron, HK Lyerly, JC Snyder A cancer rainbow mouse for visualizing the functional genomics of oncogenic clonal expansion Nat Commun, 2019-12-02;10(1):5490. 2019-12-02 [PMID: 31792216] (Cell Culture, Human)
• S Park, J Cui, WA Yu, L Wu, K Carmon, QJ Liu Differential activities and mechanisms of the four R-Spondins in potentiating Wnt/?-catenin signaling J. Biol. Chem., 2018-05-11;0(0):. 2018-05-11 [PMID: 29752411] (Bioassay, Human)
• AM Lebensohn, R Rohatgi R-spondins can potentiate WNT signaling without LGRs Elife, 2018-02-06;7(0):. 2018-02-06 [PMID: 29405118] (Bioassay, Human)
• D Zeve, DP Millay, J Seo, JM Graff Exercise-Induced Skeletal Muscle Adaptations Alter the Activity of Adipose Progenitor Cells PLoS ONE, 2016-03-25;11(3):e0152129. 2016-03-25 [PMID: 27015423] (Bioassay, Primate - Chlorocebus pygerythrus (Vervet Monkey))
• Chartier C, Raval J, Axelrod F, Bond C, Cain J, Dee-Hoskins C, Ma S, Fischer M, Shah J, Wei J, Ji M, Lam A, Stroud M, Yen W, Yeung P, Cancilla B, O'Young G, Wang M, Kapoun A, Lewicki J, Hoey T, Gurney A Therapeutic Targeting of Tumor-Derived R-Spondin Attenuates beta-Catenin Signaling and Tumorigenesis in Multiple Cancer Types. Cancer Res, 2015-12-30;76(3):713-23. 2015-12-30 [PMID: 26719531] (Bioassay, Mouse)
• Carmon K, Gong X, Yi J, Thomas A, Liu Q RSPO-LGR4 functions via IQGAP1 to potentiate Wnt signaling. Proc Natl Acad Sci U S A, 2014-03-17;111(13):E1221-9. 2014-03-17 [PMID: 24639526] (Bioassay, Human)
• Wu C, Qiu S, Lu L, Zou J, Li W, Wang O, Zhao H, Wang H, Tang J, Chen L, Xu T, Sun Z, Liao W, Luo G, Lu X RSPO2-LGR5 signaling has tumour-suppressive activity in colorectal cancer. Nat Commun, 2014-01-01;5(0):3149. 2014-01-01 [PMID: 24476626] (Bioassay, Human)
• Xie , Yang, Zamponi , Raffaell, Charlat , Olga, Ramones , Melissa, Swalley , Susanne, Jiang , Xiaomo, Rivera , Daniel, Tschantz , William, Lu , Bo, Quinn , Lisa, Dimitri , Chris, Parker , Jefferso, Jeffery , Doug, Wilcox , Sheri K, Watrobka , Mike, LeMotte , Peter, Granda , Brian, Porter , Jeffrey, Myer , Vic E, Loew , Andreas, Cong , Feng Interaction with both ZNRF3 and LGR4 is required for the signalling activity of R-spondin. EMBO Rep, 2013-10-29;14(12):1120-6. 2013-10-29 [PMID: 24165923] (Cell Culture, Human)
• Wilson TR, Fridlyand J, Yan Y, Penuel E, Burton L, Chan E, Peng J, Lin E, Wang Y, Sosman J, Ribas A, Li J, Moffat J, Sutherlin DP, Koeppen H, Merchant M, Neve R, Settleman J Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. Nature, 2012-07-26;487(7408):505-9. 2012-07-26 [PMID: 22763448] (Bioassay, Human)
• Hao HX, Xie Y, Zhang Y, Charlat O, Oster E, Avello M, Lei H, Mickanin C, Liu D, Ruffner H, Mao X, Ma Q, Zamponi R, Bouwmeester T, Finan PM, Kirschner MW, Porter JA, Serluca FC, Cong F ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature, 2012-04-29;485(7397):195-200. 2012-04-29 [PMID: 22575959] (Bioassay, Human)

Bioinformatics

• Gene Symbol: RSPO3
• Uniprot:
  • Q9BXY4(Human)