Fig. 4: RSPO2 and RSPO3 interact with BMPR1A via the TSP1 domain.
From: R-spondins are BMP receptor antagonists in Xenopus early embryonic development
a–c BRE reporter assays in HEPG2 cells transfected with constitutively active (QD) BMPR1A (a), ACVR1 (b), or BMPR1B (c) with or without BMP4 and RSPO1-4 treatment overnight. d Cell surface binding assay in HEK293T cells. (Left) Scheme of the assay. Cells were transfected with BMPR1A or LGR4 DNA, and treated with same amount of RSPO1-4-AP upon DSP crosslinking as indicated. Binding was detected as purple stain on cell surface by chromogenic AP assay. (Right) Images of cells transfected and treated as indicated. Data shows a representative from four independent experiments. For quantification, see Supplementary Fig. 5a. Scale bar, 1 mm. e In vitro binding assay between RSPO1-4, FGF and BMPR1AECD. (Left) Scheme of the assay. RSPOs and FGF recombinant proteins were coated on plate as baits, followed by BMPR1AECD-AP treatment overnight. (Right) Bound BMPR1AECD was detected by chromogenic AP assay. Normalized AP activity with control treatment was set to 1. f Scatchard plot of RSPO2 and BMPR1AECD binding to validate Kd for RSPO2-BMPR1A. g Domain structures of the RSPO1 and RSPO2TSP1 with Strep-HA and flag tags used in h. SP, signal peptide; TSP1, thrombospondin domain 1. h In vitro binding assay for RSPOTSP1 and BMPR1AECD. (Left) Scheme of the assay. HA-harboring RSPO1/2TSP1 were captured to HA antibody coated plate, and BMPR1AECD-AP was treated overnight. (Right) Bound BMPR1A to RSPOTSP1 was detected with absorbance. i Domain structures of the RSPO1, RSPO2, and R1-TSPR2. SP, signal peptide; FU, furin domain; TSP1, thrombospondin domain 1. Dashed box indicates the TSP1 domain swapping. j TOPFlash reporter assay in HEPG2 cells upon WNT3A with or without (i) as indicated. k BRE reporter assay in HEPG2 cells upon BMP4 with or without (i) as indicated. For reporter assays (a–c, j, k), n = 3 biologically independent samples; In vitro binding assays (e, h), n = 3 experimentally independent samples. All data 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 (a, b, e, h, j, k) or one-way ANOVA test (c).
