Interaction with both ZNRF3 and LGR4 is required for the signalling activity of R‐spondin

Xie, Yang; Zamponi, Raffaella; Charlat, Olga; Ramones, Melissa; Swalley, Susanne; Jiang, Xiaomo; Rivera, Daniel; Tschantz, William R.; Lü, Bo; Quinn, Lisa; Dimitri, Chris; Parker, Jefferson D.; Jeffery, D. Molkentin; Wilcox, Sheri K.; Watrobka, Mike; LeMotte, Peter K.; Granda, Brian; Porter, Jeffrey A.; Myer, Vic E.; Loew, Andreas; Cong, Feng

doi:10.1038/embor.2013.167

Cited by 93 publications

(81 citation statements)

References 25 publications

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“…A simultaneous study systematically mutating all conserved Furin repeat residues independently found that the R66A and Q71A mutations in the Furin1 domain completely abolish R-spondin-ZNRF3 binding. All studies agree that R-spondin-4 residues that are mutated in anonychia patients reside in the center of this interface Peng et al 2013b;Xie et al 2013;Zebisch et al 2013). These observations explain the inactivity of diseaserelated R-spondins in Wnt reporter assays despite intact Lgr5 binding (Peng et al 2013a;Xie et al 2013;Zebisch et al 2013).…”

Section: R-spondin Rnf43/znrf3supporting

confidence: 51%

“…Single F106E or F110E mutations in the F clamp completely abrogate receptorligand binding as well as the functional activity of R-spondin, as tested in Wnt reporter assays and small intestinal stem cell cultures (Peng et al 2013a). Similarly, F106A and F110A mutations disrupt interaction with Lgr4 (Wang et al 2013a;Xie et al 2013). A second Lgr5 interaction site in R-spondin has a charged character and centers around Lys59 (Furin-1) and Arg87 (Furin-1).…”

Section: Lgr/r-spondinmentioning

confidence: 99%

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The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength

et al. 2014

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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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Section: R-spondin Rnf43/znrf3supporting

confidence: 51%

Section: Lgr/r-spondinmentioning

confidence: 99%

The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength

et al. 2014

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“…8B). The structures and mutagenesis data (Peng et al, 2013b;Xie et al, 2013;Zebisch et al, 2013) indicated that two conserved RSPO residues are most critical for ZNRF3/RNF43 binding: R66 and Q71 (RSPO1 numbering), which are located on the b-strands of the second b-hairpin of Fu1 and face into a shallow cleft on the ZNRF3/RNF43 surface (Figs. 4B and 8A).…”

Section: Resultsmentioning

confidence: 99%

“…LGR4/5/6 have a large extracellular domain (ECD) with 17 leucine-rich repeats (LRR) that provide the RSPO binding site, and ZNRF3/RNF43 have a small ECD for RSPO binding. Crystal structures of binary and ternary complexes Peng et al, 2013a,b;Wang et al, 2013;Xu et al, 2013;Zebisch et al, 2013) and mutagenesis studies (Xie et al, 2013) indicated that distinct regions of RSPO Fu1-Fu2 contact the two receptors.…”

Section: Introductionmentioning

confidence: 99%

Engineering High-Potency R-spondin Adult Stem Cell Growth Factors

2014

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Secreted R-spondin proteins (RSPOs1-4) function as adult stem cell growth factors by potentiating Wnt signaling. Simultaneous binding of distinct regions of the RSPO Fu1-Fu2 domain module to the extracellular domains (ECDs) of the LGR4 G protein-coupled receptor and the ZNRF3 transmembrane E3 ubiquitin ligase regulates Wnt receptor availability. Here, we examine the molecular basis for the differing signaling strengths of RSPOs1-4 using purified RSPO Fu1-Fu2, LGR4 ECD, and ZNRF3 ECD proteins in Wnt signaling and receptor binding assays, and we engineer novel high-potency RSPOs. RSPO2/3/4 had similar signaling potencies that were stronger than that of RSPO1, whereas RSPO1/2/3 had similar efficacies that were greater than that of RSPO4. The RSPOs bound LGR4 with affinity rank order RSPO4 . RSPO2/3 . RSPO1 and ZNRF3 with affinity rank order RSPO2/3 . . RSPO1 .RSPO4. An RSPO2-4 chimera combining RSPO2 ZNRF3 binding with RSPO4 LGR4 binding was a "Superspondin" that exhibited enhanced ternary complex formation and 10-fold stronger signaling potency than RSPO2 and efficacy equivalent to RSPO2. An RSPO4-1 chimera combining RSPO4 ZNRF3 binding with RSPO1LGR4 binding was a "Poorspondin" that exhibited signaling potency similar to RSPO1 and efficacy equivalent to RSPO4. Conferring increased ZNRF3 binding upon RSPO4 with amino acid substitutions L56F, I58L, and I63M enhanced its signaling potency and efficacy. Our results reveal the molecular basis for RSPOs1-4 activity differences and suggest that signaling potency is determined by ternary complex formation ability, whereas efficacy depends on ZNRF3 recruitment. High-potency RSPOs may be of value for regenerative medicine and/or therapeutic applications.

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“…RSPOs normally inhibit the activity of RNF43 and ZNRF3 by sequestering them into a heterotrimeric complex with LGR4/5/6 (Leucine-rich repeat containing G-protein-coupled receptors 4, 5, or 6). This complex results in the inhibition and membrane clearance of these E3 ubiquitin ligases, leading to increased Frizzled and LRP5/6 proteins on the cell surface (43,54). The LGR RSPO complex may also enhances b-catenin-dependent signaling by promoting MEK1/2-mediated phosphorylation of LRP5/6 and b-catenin-independent signaling through regulation of actin dynamics (55).…”

Section: R-spondin Translocations and Gene Amplificationmentioning

confidence: 99%

Targeting Wnts at the Source—New Mechanisms, New Biomarkers, New Drugs

Madan

Virshup

2015

Molecular Cancer Therapeutics

103

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Wnt signaling is dysregulated in many cancers and is therefore an attractive therapeutic target. The focus of drug development has recently shifted away from downstream inhibitors of b-catenin. Active inhibitors of Wnt secretion and Wnt/receptor interactions have been developed that are now entering clinical trials. Such agents include inhibitors of Wnt secretion, as well as recombinant proteins that minimize Wnt-Frizzled interactions. These new therapies arrive together with the recent insight that cancer-specific upregulation of Wnt receptors at the cell surface regulates cellular sensitivity to Wnts. Loss-of-function mutations in RNF43 or ZNRF3 and gain-of-function chromosome translocations involving RSPO2 and RSPO3 are surprisingly common and markedly increase Wnt/b-catenin signaling in response to secreted Wnts. These mutations may be predictive biomarkers to select patients responsive to newly developed upstream Wnt inhibitors.

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Interaction with both ZNRF3 and LGR4 is required for the signalling activity of R‐spondin

Cited by 93 publications

References 25 publications

The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength

The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength

Engineering High-Potency R-spondin Adult Stem Cell Growth Factors

Targeting Wnts at the Source—New Mechanisms, New Biomarkers, New Drugs

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