Extended Data Fig. 2: The RSPO2(R69C) and RSPO2(Q70X) mutants fail to bind ZNRF3.
From: RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6
a, The RSPO2 R69 residue (highlighted in purple) is highly conserved in vertebrates and within the human paralogues RSPO1–RSPO4. Conserved cysteine residues of the Furin-like 1 domain are highlighted in pink. Protein alignment performed with ClustalO. b, Western blotting of protein extracts and supernatants from HEK293T cells transfected with indicated constructs. Deletion of the RSPO2 C-terminal domain (RSPO2-ΔC) decreases its retention on the cell surface without affecting its receptor binding and WNT enhancement properties38. The RSPO2(R69C) mutant was almost undetectable in conditioned media but was greatly increased by the addition of heparin in the medium. c, Co-immunoprecipitation of wild-type and mutant forms of RSPO2-ΔC-AP with the ProteinG-Flag beads only. Asterisk indicates an unspecific band. d, Co-immunoprecipitation of wild-type and mutant forms of RSPO2-ΔC-AP with the ZNRF3-ECD-Flag E3 ligase. Asterisk indicates an unspecific band. e, Cell-surface binding assay of HEK293T cells transfected with empty vector, LGR5 or RNF43, using equivalent amounts of RSPO2-ΔC-AP conditioned media (western blot). Experiments in b–e were repeated three times. f, SUPERTOPFLASH assay in HEK293T-STF cells transfected with WNT3A in the presence of equivalent amounts of RSPO2-ΔC-AP conditioned media (western blot). n = 4 biological replicates. Data are mean ± s.e.m. NS, not significant. **P < 0.01, one-way ANOVA test with Bonferroni’s correction. For gel source data, see Supplementary Fig. 1.
