Fig. 8: RSPO2 bridges BMPR1A and ZNRF3 and triggers BMP receptor clearance from the cell surface.
From: R-spondins are BMP receptor antagonists in Xenopus early embryonic development
a, b In vitro binding assay between ZNRF3 and BMPR1AECD mediated by RSPO1-3. ZNRF3-Fc protein was used as a bait, with sequential RSPO1-3 protein and BMPR1AECD-AP treatment. Bound BMPR1AECD to ZNRF3 was detected by chromogenic AP assay. n = 3 experimentally independent samples. c, d IF in H1581 cells transfected with BMPR1A-HA and ZNRF3-flag DNA upon RSPO2 and RSPO3-HRP treatment for 3 h. RSPOs (red) were visualized with tyramid signal amplification. BMPR1A (blue) and ZNRF3 (green) were stained against HA and flag antibody. White arrowheads, colocalized BMPR1A/RSPO2; white arrows, colocalized BMPR1A/RSPO2-3/ZNRF3; yellow arrow, colocalized BMPR1A/RSPO2-3/ZNRF3 in magnified inset; Dashed lines, nucleus. Scale bar, 20 μm. e–h IF of colocalized BMPR1A (green)/ZNRF3 (red) in H1581 cells treated with siRNA as indicated. Nuclei were stained with Hoechst. Scale bar, 20 μm. i Quantification of BMPR1A colocalizing with ZNRF3 from e–h. j–l IF of BMPR1A (green) in H1581 cells treated with siRNA as indicated. m Quantification of cells harboring membrane localized BMPR1A from j–l. Scale bar, 20 μm. n Cell surface biotinylation assay in H1581 cells treated with BMPR1A-HA and siRNA as indicated. Co, control; R2, RSPO2; ZR, ZNRF3/RNF43 siRNA. After labeling surface proteins with Biotin, lysates were pulled down with streptavidin beads and subjected to Western blot analysis. Transferrin receptor (TfR), a loading control. TCL, Total cell lysate. Data shows results representative for 2 from 3 independent experiments. o BRE reporter assay in HEPG2 cells treated as indicated. MDC, monodansylcadaverin. n = 3 biological replicates. p IF of colocalized BMPR1A (green)/EEA1 (magenta) in H1581 cells treated with siRNA. White arrowheads, colocalized BMPR1A/EEA1 in magnified inset. q Quantification of p. Scale bar, 20 μm. r IF of colocalized BMPR1A (green)/Lamp1 (red) in H1581 cells treated with siRNA as indicated. White arrowheads, colocalized BMPR1A/Lamp1 in magnified inset. Scale bar, 20 μm. s Quantification of r. For i, m, q, s, n = the number of cells pooled from 2 independent experiments. Data for (a, b, i, m, o, q, s) are displayed as mean ± SD. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 from two-tailed unpaired t-test.
