Loss-of-function point mutations and two-furin domain derivatives provide insights  about R-spondin2 structure and function.
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.
Li
Sheng-Jian
SJ
Laboratory of Cellular and Molecular Biology, National Cancer Institute, 37 Convent Drive, Bethesda, MD 20892, USA.
Yen
Ten-Yang
TY
Endo
Yoshimi
Y
Klauzinska
Malgorzata
M
Baljinnyam
Bolormaa
B
Macher
Bruce
B
Callahan
Robert
R
Rubin
Jeffrey S
JS
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides; D045683:Furin / Q000737:chemistry*; D017930:Genes, Reporter; D006801:Humans; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D017354:Point Mutation / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000378:metabolism; D013329:Structure-Activity Relationship; D019699:Thrombospondins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism; D000098907:R-Spondins
D003545:Cysteine; D004220:Disulfides; D045683:Furin; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D050505:Mutant Proteins; D010455:Peptides; D011973:Receptors, LDL; D019699:Thrombospondins; D051176:beta Catenin; C555475:LRP6 protein, human; C555476:Lrp6 protein, mouse; C518904:RSPO2 protein, mouse; D000098907:R-Spondins