Fig 1.
The R-spondin signaling mechanism.
In the absence of RSPO, ZNRF3 drives membrane clearance of Fzd receptors to negatively regulate Wnt signaling. RSPO-mediated crosslinking of the ECDs of RNF43 or ZNRF3 and LGR4, LGR5, or LGR6 sequesters RNF43/ZNRF3 to restore Fzd surface levels, thereby potentiating Wnt activity. Surrogate RSPOs mimic the function of wild type RSPOs by cross-linking RNF43 or ZNRF3 to tissue-specific markers known to undergo endocytosis upon ligand binding. PDB files 5UK5[7], 4F0A[19], 3S8Z, and 3S94[20] were used to generate the images above.
Fig 2.
Characterization of RNF43- and ZNRF3-specific scFvs used for generation of surrogate RSPOs.
(a) Cartoon schematic of yeast-displayed scFv binding to biotinylated RNF43 or ZNRF3 ECDs. (b) Flow cytometry dot plots depicting the binding of R5 and Z6 scFvs to RNF43 and ZNRF3, respectively. R5- or Z6-expressing yeast were stained with 1 μM concentrations of biotinylated RNF43 or ZNRF3 ECDs, respectively, and binding was detected using fluorescently labeled streptavidin. Surface expression of R5 and Z6 was detected with an antibody to the c-Myc epitope. Negative controls are unstained R5- or Z6-expressing yeast. (c) Construct design for R5-IL2 and Z6-IL2 surrogate RSPOs. (d) & (e). SPR was used to measure the binding of R5-IL2 or Z6-IL2 to RNF43 and ZNRF3, respectively. Cartoons (left) depict the orientation of streptavidin (SA)-coupled RNF43 or ZNRF3 ECDs and the R5-IL2 or Z6-IL2 analytes on the sensor chip. Curves (center) indicate a series of injections of R5-IL2 (3-fold dilutions, maximum concentration 3 μM) or Z6-IL2 (2-fold dilutions, maximum concentration 1 μM). Dissociation constants were obtained by plotting maximum RU values and fitting to a 1:1 binding model (right).
Fig 3.
Surrogate RSPOs potentiate Wnt signaling.
(A) Representative Luciferase reporter assay measuring surrogate RSPO potentiation of Wnt activity in CD25-expressing cells. CD25-expressing HEK STF 293T cells were treated 20% WNT3a conditioned media and various recombinant proteins (RSPO2 Furin domains 1 and 2 (25 nM), R5 (500 nM), Z6 (500 nM), IL-2 (500 nM), R5-IL2 (5 nM, 50 nM, 500 nM), Z6-IL2 (5nM, 50 nM, 500 nM), or a 1:1 mixture of R5-IL2 and Z6-IL2 (5 nM each, 50 nM each, 500 nM each). The Luciferase signal fold change is normalized to WNT3a alone. Error bars represent standard deviation of n = 3 technical replicates. Statistical significance of WNT3a alone versus each condition was determined by using one-way ANOVA on log-transformed data. * P<0.05, ** P<0.01, *** P<0.001. (B) A reporter assay was performed under the same conditions as in (A) using uninfected (CD25 negative) HEK STF 293T cells. * P<0.05, ** P<0.01, *** P<0.001.
Fig 4.
Surrogate RSPOs stimulate intestinal organoid growth.
Surrogate RSPOs were tested for their ability to stimulate the growth of LGR5+ human colon organoids that had been retrovirally transduced to express CD25. Basal media containing WNT3a, EGF and Noggin, but lacking RSPO2 was supplemented with 500nM concentrations of the indicated proteins, or with 25nM RSPO2. Microscopy was used to observe the appearance of spherical organoids in each condition (top) and growth was quantitated by resazurin fluorescence in a cell viability assay (bar graphs, bottom). The bar graph on the right is a zoomed panel of the colored region from the graph on the left. Error bars represent standard error of n = 12.