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

Abstract

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.

An anterior signaling center patterns and sizes the anterior neuroectoderm of the sea urchin embryo

“…sFRPs comprise the largest family of Wnt modulators in the animal kingdom (Bovolenta et al, 2008). They contain a cysteine-rich domain (CRD) that is almost identical to the CRD of the Wnt receptor Frizzled (Fzl), and several studies suggest that sFRPs could bind Wnt via this domain and inhibit the ability of Wnt to activate the Fzl receptor (Bafico et al, 1999;Bhat et al, 2007;Lin et al, 1997 (Bafico et al, 1999;Dufourcq et al, 2008;Esteve et al, 2011;Taira, 2009, 2011;Rodriguez et al, 2005). Based on these apparently conflicting results, it has been proposed that the functions of sFRPs depend on their expression levels as well as the molecular and cellular context (Bovolenta et al, 2008;Mii and Taira, 2011).…”

Sizzled Is Unique among Secreted Frizzled-related Proteins for Its Ability to Specifically Inhibit Bone Morphogenetic Protein-1 (BMP-1)/Tolloid-like Proteinases

“…Most importantly also, we have shown that both conserved and non-conserved residues were required to allow the inhibitory complex with BMP-1 to form, explaining why sFRP-1 and -2 cannot form "productive" complexes with BMP-1. Interestingly, Ser-43 and Glu-44 in sizzled align with the KK motif in the short sequence 74 YKKM of sFRP-1, which has been shown to be necessary for Wnt3a antagonist activity (67), pointing to the important functional role played by this loop.…”

Secreted and Transmembrane Wnt Inhibitors and Activators

“…In contrast, a mutant of sFRP1 lacking the CRD domain retained the ability to bind to Wingless, the Drosophila Wnt homolog (Uren et al 2000). Another study of sFRP1 structure and function using Wnt reporter assays indicates that both protein domains, CRD and NTR, are important for optimal Wnt inhibition (Bhat et al 2007). Furthermore, a recent study on sFRP1 combining biochemical and functional assays in cell culture and medakafish embryos shows that its NTR domain mimics the function of the entire molecule in binding Wnt8 and inhibiting Wnt signaling .…”