Structure-Function analysis of secreted frizzled-related protein-1 for its Wnt antagonist function

Secreted frizzled‐related proteins (sFRPs) are glycoproteins that are recognized as Wnt antagonists. To identify the functional domains that are involved in Wnt antagonist function, several sFRP‐1 mutants and sFRP‐1/sFRP‐2 chimeras were generated. These mutants were characterized in an optimized T‐cell factor (TCF)‐luciferase based assay in U2OS human osteosarcoma cells. Deletions of the sFRP‐1 cysteine rich domain (CRD) lead to the complete loss of Wnt antagonist function. A region between amino acids 73–86 within the second loop of the CRD of sFRP‐1 was necessary for the optimal Wnt inhibitory function. Within this region, a conserved tyrosine residue played a critical role, and its change to neutral or polar amino acids lead to decreased Wnt inhibitory activity. The sFRP‐1/sFRP‐2 chimeras with the netrin domain of sFRP‐1 replaced by corresponding sFRP‐2 sequences showed 40–70% loss of Wnt antagonist function. The sFRP‐1/sFRP‐2 chimera with the replacement of C‐terminal 19 amino acids of sFRP‐1 with 11 amino acids of sFRP‐2 resulted in 70% loss of activity indicating that carboxyl‐terminal region of sFRP‐1 is important for its Wnt inhibitory activity. The structure–function analysis studies of sFRP‐1 clearly demonstrate the interaction of several functional domains for its optimal Wnt antagonist function. J. Cell. Biochem. 102: 1519–1528, 2007. © 2007 Wiley‐Liss, Inc.