Secreted Frizzled-related Protein-1 Binds Directly to Wingless and Is a Biphasic Modulator of Wnt Signaling

Abstract

Secreted Frizzled-related protein-1 (sFRP-1) contains a cysteine-rich domain homologous to the putative Wntbinding site of Frizzleds. To facilitate the biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields ϳ3 mg of purified protein/liter of conditioned medium. Using this recombinant protein, we demonstrated that sFRP-1 and Wg (wingless) interact in enzyme-linked immunosorbent and co-precipitation assays. Surprisingly, a derivative lacking the cysteine-rich domain retained the ability to bind Wg. Cross-linking experiments performed with radioiodinated sFRP-1 provided definitive evidence that sFRP-1 and Wg bind directly to each other. Besides detecting a cross-linked complex consistent in size with 1:1 stoichiometry of sFRP-1 and Wg, we also observed a larger complex whose size suggested the presence of a second sFRP-1 molecule. The formation of both complexes was markedly enhanced by an optimal concentration of exogenous heparin, emphasizing the potential importance of heparan-sulfate proteoglycan in Wnt binding and signaling. sFRP-1 exerted a biphasic effect on Wg activity in an armadillo stabilization assay, increasing armadillo level at low concentrations but reducing it at higher concentrations. These results provide new insights about the Wnt binding and biological activity of sFRPs.

Regulation of fibronectin and metalloproteinase expression by Wnt signaling in rheumatoid arthritis synoviocytes

“…Several lines of evidence suggest that because of considerable homology to the extracellular domain of the Fz receptors, high concentrations of sFRP-1 can block the interactions of Wnt-1 and Fz (32,33). The RA FLS we used for our experiments did not express much sFRP-1, either at the message level or at the protein level (data not shown).…”

“…The dnTCF-4-HA and full-length ␤-catenin expression constructs were kind gifts of Dr. Hans Clevers (University Medical Center, Utrecht, The Netherlands). The sFRP-1 expression vector has been described previously (32,33). The dnLEF-1 construct was made by amplifying the human LEF-1 sequence without the ␤-catenin binding domain and cloning the amplified product into the expression vector pcDNA3.…”

“…Recombinant sFRP-1 protein was purified from conditioned medium of MDCK cells transfected with the sFRP-1 expression vector as described previously (32,33). The recombinant protein, dissolved in phosphate buffered saline (PBS) containing 1M NaCl was added to cultures of RA FLS that had been plated a day before in 6-well plates, at a density of ϳ2 ϫ 10 5 cells/ml.…”

Molecular regulation of kidney development: is the answer blowing in the Wnt?

“…A third possibility is that reporter activation is Wnt independent. Recent data suggest that a number of non-Wnt factors are able to activate the β-catenin pathway, including the secreted molecules norrin, dickopf 2, Sfrp1 and the R-spondins [72][73][74][75][76]. Although several of these molecules are expressed in the embryonic kidney, their roles in the development of this organ are unclear, although sFrp-1 has been suggested to have an inhibitory role in tubule formation [77].…”

Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer

“…Notably, SFRP4 is less frequently hypermethylated in colorectal cancer 5 and has the least homology with other family members 14 . SFRP1 is thought to silence ligand-dependent WNT signaling by binding of the cysteine rich-domain (CRD) to WNT proteins, thus preventing interaction with FRZ receptors [15][16][17][18] . When we transfected SFRP2 deletion mutants lacking either the cysteine-rich domain (CRD) or the netrin-like domain ( Fig.…”