Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function

Abstract

R-spondins (Rspos) potentiate Wnt/beta-catenin signaling, an important pathway in embryonic development that is constitutively active in many cancers. To analyze Rspo structure and function, we expressed full-length wild-type Rspo2 and Rspo2 point mutants corresponding to Rspo4 variants that have been linked to developmental defects. The Rspo2 mutants had markedly reduced potency relative to the wild-type protein,demonstrating for the first time specific amino acid residues in Rspos that are critical for beta-catenin signaling. The diminished activity of Rspo2/C78Y and Rspo2/C113R was attributable to a defect in their secretion, while Rspo2/Q70R exhibited a decrease in its intrinsic activity. Cysteine assignments in a Rspo2 derivative containing only the two furin-like domains (Rspo2-2F) provided the first information about the disulfide bonding pattern of this motif, which was characterized by multiple short loops and unpaired cysteine residues, and established that the loss-of-function cysteine mutants disrupted disulfide bond formation. Moreover, Rspo2-2F demonstrated potent activity and synergized strongly with Wnt-3a in a beta-catenin reporter assay. In contrast, an Rspo2-2F derivative containing the Q70R substitution showed significantly reduced activity, although it still synergized with Wnt-3a in the reporter assay. Rspo2-2F derivatives elicited an unusually sustained phosphorylation (20 h) of the Wnt co-receptor, low density lipoprotein receptor-related protein 6 (LRP6), as well as an increase in cell surface LRP6. Co-immunoprecipitation experiments involving LRP6 and Kremens suggested that these associations contribute to Rspo2 activity, although the lack of major differences between wild-type and Q70R derivatives implied that additional interactions may be important.

Figure 1. Rspo2/C78Y, Rspo2/C113R and Rspo2/Q70R mutants had markedly reduced activity in Wnt/β-catenin pathway

(A) Luciferase reporter assay. STF cells were transfected with 6 μg of empty vector or indicated Rspo2 construct and lysed 48 h later for luminescence measurements. Values were normalized according to total protein concentration of cell lysates, and luciferase activity was expressed as fold stimulation relative to results with the empty vector. Lysates were assayed in triplicate; error bars (often not visible in figure) represent S.D. (B) Immunoblot analysis of Rspo2 derivatives, β-catenin and LRP6 in transfected cells. After 48 h, lysates were prepared from transfected cells and blotted for β-catenin, phospho-LRP6, total LRP6, Rspo2 derivatives and HSP70 as a loading control. Free β-catenin was detected after pull-down with GST-E-cadherin. Rspo2 proteins also were visualized in concentrated CM. Note that the entire CM from transfected monolayer cultures were loaded on the gel, whereas only 1/15 of cell lysates were subjected to western blotting.

Figure 2. Rspo2 cysteine mutants exhibited defective secretion in CHO cell culture

CHO cells transiently transfected with empty vector or the indicated Rspo2 construct were fixed and stained under non-permeabilization conditions to visualize Rspo2 proteins on the cell surface or in the extracellular space. Rspo2 proteins were detected with V5 antibody, while cell boundaries were indicated by phalloidin staining of polymerized actin and DAPI stained nuclei.

Figure 3. Characterization of purified Rspo2-2F and Rspo2-2F/Q70R proteins

(A) Coomassie blue staining of purified Rspo2-2F and Rspo2-2F/Q70R proteins (700 ng/lane). (B) Western blot of purified Rspo2-2F and Rspo2-2F/Q70R proteins (50 ng/lane) with anti-Rspo2 antibody. (C) Circular dichroism spectrum of nickel-purified Rspo2-2F. The circular dichroism spectrum was collected using a Jasco J720 spectropolarimeter, with a 30 μM protein solution in PBS. (D) Amino acid sequence and cysteine assignments of Rspo2-2F. The sequence of the purified protein is shown in standard font; the italicized segment was upstream sequence removed by Kex2 cleavage. Lines between cysteines indicate disulfide linkages and asterisks highlight free cysteines; the status of underlined cysteines was not been determined. Numbering corresponds to the sequence of full-length mouse Rspo2.

Figure 4. Comparison and contrast of Rspo2-2F and Rspo2-2F/Q70R activity in β-catenin pathway

(A) Luciferase reporter activity of purified Rspo2-2F (◆) or Rspo2-2F/Q70R (■) at different concentration (10, 30, 100 and 300 ng/ml). (B) Luciferase reporter activity of purified Rspo2-2F (100 ng/ml), Rspo2-2F/Q70R (100 ng/ml) or Wnt-3a (50 ng/ml) alone or in combination. STF cells were treated with purified proteins for 20 h in serum-free DMEM and luminescence was normalized to total protein concentration of cell lysates. Relative luciferase activity was defined as the fold stimulation of data obtained from cells treated with PBS. (C) Time course analysis of LRP6 phosphorylation and total LRP6 content in cells treated with Rspo2-2F, Rspo2-2F/Q70R or Wnt-3a alone or in combination. Cells were incubated for 1, 6 or 20 h with the indicated factors prior to processing for immunoblotting. (D) Biotinylated and total LRP6 protein in cells treated with Rspo2-2F, Rspo2-2F/Q70R or Wnt-3a alone or in combination. Following 20 h incubation with the indicated factors, intact cells were subjected to biotinylation and biotinylated proteins were pelleted with streptavidin-conjugated beads. LRP6 recovered in these pellets and LRP6 present in whole cell lysates (50 μg/lane) were detected by immunoblotting. HSP70 detection served as a loading control for the cell lysates in (C) and (D). The numbers above lanes in (D) indicate band signal intensities, after normalization to the corresponding HSP70 signal, relative to the zero time point from one of three experiments with similar results.

Figure 5. Rspo2 and Rspo2/Q70R association with LRP6

(A) Co-expression of V5-tagged Rspo2, Rspo2/Q70R and Dkk1 with LRP6-Myc₆ in lysates from transiently transfected HEK293 cells. (B) Co-IP of V5-tagged proteins with LRP6-Myc₆, using Myc antibody or control mouse IgG. Precipitates were immunoblotted in parallel with V5 and Myc antibodies. (C) Co-IP of V5-tagged proteins with LRP6-Myc₆, using V5 antibody or control mouse IgG. Precipitates were immunoblotted in parallel with Myc and V5 antibodies.

Figure 6. Rspo2 and Rspo2/Q70R association with Krm2

(A) Co-expression of V5-tagged Rspo2, Rspo2/Q70R and Dkk1 with Krm2-FLAG in lysates from transiently transfected CHO cells. (B) Co-IP of V5-tagged proteins with Krm2-FLAG using FLAG antibody or control mouse IgG. Precipitates were immunoblotted in parallel with V5 and FLAG antibodies. (C) Co-IP of V5-tagged proteins with Krm2-FLAG, using V5 antibody or control mouse IgG. Precipitates were immunoblotted in parallel with FLAG and V5 antibodies.