Secreted frizzled-related protein-1 binds directly to wingless and is a biphasic modulator of Wnt signaling

Secreted Frizzled-related protein-1 (sFRP-1) contains a cysteine-rich domai n homologous to the putative Wnt-binding site of Frizzleds. To facilitate t he biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields similar to 3 mg of purified prote in/liter of conditioned medium. Using this recombinant protein, we demonstr ated that sFRP-1 and Wg (wingless) interact in enzyme-linked immunosorbent and co-precipitation assays. Surprisingly, a derivative lacking the cystein e-rich domain retained the ability to bind Wg, Cross-linking experiments pe rformed with radioiodinated sFRP-1 provided definitive evidence that sFRP-1 and Wg bind directly to each other. Besides detecting a cross-linked compl ex consistent in size with 1:1 stoichiometry of sFRP-1 and Wg, we also obse rved a larger complex whose size suggested the presence of a second sFRP-1 molecule. The formation of both complexes was markedly enhanced by an optim al concentration of exogenous heparin, emphasizing the potential importance of heparan-sulfate proteoglycan in Wnt binding and signaling. sFRP-1 exert ed a biphasic effect on Wg activity in an armadillo stabilization assay, in creasing armadillo level at low concentrations but reducing it at higher co ncentrations. These results provide new insights about the Wnt binding and biological activity of sFRPs.