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. 2013 Dec;14(12):1120-6.
doi: 10.1038/embor.2013.167. Epub 2013 Oct 29.

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

Yang Xie  1 , Raffaella Zamponi, Olga Charlat, Melissa Ramones, Susanne Swalley, Xiaomo Jiang, Daniel Rivera, William Tschantz, Bo Lu, Lisa Quinn, Chris Dimitri, Jefferson Parker, Doug Jeffery, Sheri K Wilcox, Mike Watrobka, Peter LeMotte, Brian Granda, Jeffrey A Porter, Vic E Myer, Andreas Loew, Feng Cong
Affiliations

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

Yang Xie et al. EMBO Rep. 2013 Dec.

Abstract

R-spondin proteins sensitize cells to Wnt signalling and act as potent stem cell growth factors. Various membrane proteins have been proposed as potential receptors of R-spondin, including LGR4/5, membrane E3 ubiquitin ligases ZNRF3/RNF43 and several others proteins. Here, we show that R-spondin interacts with ZNRF3/RNF43 and LGR4 through distinct motifs. Both LGR4 and ZNRF3 binding motifs are required for R-spondin-induced LGR4/ZNRF3 interaction, membrane clearance of ZNRF3 and activation of Wnt signalling. Importantly, Wnt-inhibitory activity of ZNRF3, but not of a ZNRF3 mutant with reduced affinity to R-spondin, can be strongly suppressed by R-spondin, suggesting that R-spondin primarily functions by binding and inhibiting ZNRF3. Together, our results support a dual receptor model of R-spondin action, where LGR4/5 serve as the engagement receptor whereas ZNRF3/RNF43 function as the effector receptor.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
RSPO1 interacts with LGR4 and ZNRF3 through distinct motifs. (A) A schematic diagram of the domain structure of human RSPO1. (B) Differential binding of RSPO1–GFP mutants to LGR4 and ZNRF3 ECD-TM in a cell-based binding assay. HEK293 cells transiently overexpressing LGR4 or ZNRF3 ECD–TM were incubated with RSPO1–GFP conditioned medium (CM), and binding of RSPO1–GFP was analysed by fluorescence microscopy. Similar results were obtained in three independent experiments. (C) Interaction between WT RSPO1–GFP, R66A/Q71A double mutant or F106A/F110A double mutant and RNF43–ECD-TM in cell-based binding assay. (D) The level of wild-type or mutant RSPO1–GFP used in cell-based binding assay. The level of wild-type or mutant RSPO1–GFP in conditioned medium was determined by western blot using anti-GFP antibody. ECD, extracellular domain; FU1, furin-like domain 1; FU2, furin-like domain 2; GFP, green fluorescent protein; SP, signal peptide; TSP, thrombospondin domain.
Figure 2
Figure 2
Interaction between RSPO1 and ZNRF3 ECD in surface plasmon resonance assay. (A) Interaction between RSPO1 and ZNRF3 ECD. (B) Interaction between RSPO1 R66A and ZNRF3 ECD. (C) Interaction between RSPO1 and ZNRF3 ECD P103A. ECD, extracellular domain.
Figure 3
Figure 3
Physical interaction with both LGR4 and ZNRF3 is required for the signaling activity of R-spondin. (A) RSPO1–GFP mutants with defective LGR4 or ZNRF3 binding lose Wnt-potentiating activity. HEK293–STF cells were treated with wild type or mutants of RSPO1–GFP in the presence of control conditioned medium (L CM) or 1% Wnt3a CM, and STF luciferase reporter activity was measured. Note that RSPO1 promotes the signalling activity of endogenous Wnt proteins in HEK293 cells. The same amount of RSPO1–GFP was used in the experiment. Error bars denote the s.d. Data are representative of three independent experiments. (B) RSPO2–GFP FU1 or FU2 double mutants have compromised Wnt-stimulating activity. The amount of wild type or mutants RSPO2–GFP was examined by anti-GFP immunoblotting. Data are representative of three independent experiments. (C) RSPO3–GFP FU1 or FU2 double mutants have compromised Wnt-stimulating activity. The amount of wild type or mutants RSPO3–GFP was examined by anti-GFP immunoblotting. Data are representative of three independent experiments. (D) Wild-type but not mutant RSPO1–GFP induces the association between LGR4-HA and Myc-ZNRF3ΔRING. HEK293 cells co-expressing indicated plasmids were treated with RSPO1–GFP conditioned medium for 1 h. Cell lysates were immunoprecipitated with anti-Myc antibody, and immunoprecipitates were resolved and blotted with indicated antibodies. (E) Wild-type but not mutant RSPO1–GFP induces membrane clearance of ZNRF3. HEK293 cells co-expressing LGR4 and Myc-ZNRF3 were treated with wild-type or mutant ZNRF3 for 24 h. The level of Myc-ZNRF3 on the cell surface was quantified by FACS. (F) RSPO1 R66A/Q71A shows dominant negative activity. HEK293–STF cells were treated with wild-type RSPO1–GFP with or without 10-fold excess of indicated RSPO1–GFP mutant. Data are representative of three independent experiments. Statistical significance was assessed by Student’s t-test. *P<0.01. CM, conditioned medium; FU1, furin-like domain 1; FU2, furin-like domain 2; GFP, green fluorescent protein; STF, SuperTopflash.
Figure 4
Figure 4
Characterization of RSPO1 SOMAmer. (A) RSPO1 SOMAmer specifically inhibits RSPO1-induced STF. HEK293–STF cells were treated with 4 nM RSPO1 plus 5% Wnt3a CM, 4 nM RSPO3 plus 5% Wnt3a CM or 40% Wnt3a alone, together with SOMAmer at indicated concentrations. After overnight treatment, STF luciferase activity was measured. S1, RSPO1-specific SOMAmer. Ctr, scrambled control. Data are representative of three independent experiments. (B) RSPO1 SOMAmer has defective binding to RSPO1 R66A/Q71A mutant. Conditioned medium of RSPO1–GFP WT, R66A/Q71A or F106A/F110A was incubated with biotinylated RSPO1-specific SOMAmer (S1) or scrambled control (Ctr). RSPO1–GFP pulled down by SOMAmers was analysed by immunoblotting using anti-GFP antibody. (C) RSPO1-specific SOMAmer blocks the interaction between RSPO1-Fc and ZNRF3 or LGR4. SOMAmer S1 or scrambled control (Ctr) was incubated with RSPO1-Fc and HEK293 cells overexpressing LGR4 or ZNRF3, and RSPO1-Fc binding to the receptors was detected by immunofluorescence microscopy. CM, conditioned medium; ECD, extracellular domain; EV, empty vector; GFP, green fluorescent protein; SOMAmer, slow off-rate modified aptamers; STF, SuperTopflash; WT, wild type.
Figure 5
Figure 5
Cells expressing ZNRF3 mutant with defective R-spondin interaction are refractory to the Wnt-stimulatory activity of R-spondin. (A) HEK293 cells were first transfected with control pGL2 siRNA or ZNRF3 siRNA to eliminate endogenous ZNRF3. Twenty-four hours after siRNA transfection, cells were transfected with empty vector (EV), Myc-ZNRF3 WT or Myc-ZNRF3 H102A/ P103A expression plasmid, and STF reporter. Twenty-four hours after plasmid transfection, all wells were treated with 5% Wnt3a and selected wells were treated with 200 ng/ml of RSPO2 proteins. STF luciferase activity was measured 24 h later. Data are representative of three independent experiments. (B) ZNRF3 WT and ZNRF3 H102A/P103A are expressed on the cell surface and total cell lysates (TCL) at the similar level. Cells transfected with Myc-tagged ZNRF3 WT or ZNRF3 H102A/P103A were subjected to membrane biotinylation, and biotinylated membrane proteins were pulled down with avidin beads, fractionated and blotted with anti-Myc antibody. CM, conditioned medium; ECD, extracellular domain; EV, empty vector; GFP, green fluorescent protein; siRNA, small interfering RNA; STF, SuperTopflash; TCL, total cell lysates; WT, wild type.
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