Purification and molecular cloning of a secreted, Frizzled-related antagonist of Wnt action

Purification and molecular cloning of a secreted, Frizzled-related antagonist of Wnt action

^*Derald H. Ruttenberg Cancer Center, Mount Sinai Medical Center, New York, NY 10029-6574; and ^†Varmus Laboratory, ^§Medicine Branch, and Laboratories of ^¶Cellular and Molecular Biology, ^**Experimental Carcinogenesis, and ^‡‡Genetics, National Cancer Institute, Bethesda, MD 20892

Abstract

Frizzled polypeptides are integral membrane proteins that recently were shown to function as receptors for Wnt signaling molecules. Here, we report the identification of a novel, secreted 36-kDa protein that contains a region homologous to a putative Wnt-binding domain of Frizzleds. This protein, called Frizzled-related protein (FRP), was first identified as a heparin-binding polypeptide that copurified with hepatocyte growth factor/scatter factor in conditioned medium from a human embryonic lung fibroblast line. Degenerate oligonucleotides, based on the NH₂-terminal sequence of the purified protein, were used to isolate corresponding cDNA clones. These encoded a 313-amino acid polypeptide, containing a cysteine-rich domain of ≈110 residues that was 30–40% identical to the putative ligand-binding domain of Frizzled proteins. A 4.4-kb transcript of the FRP gene is present in many organs, both in the adult and during embryogenesis, and homologs of the gene are detectable in DNA from several vertebrate species. In biosynthetic studies, FRP was secreted but, like Wnts, tended to remain associated with cells. When coexpressed with several Wnt family members in early Xenopus embryos, FRP antagonized Wnt-dependent duplication of the embryonic dorsal axis. These results indicate that FRP may function as an inhibitor of Wnt action during development and in the adult.

Footnotes

↵ ‡ Present address: Division of Neuroscience, Children’s Hospital/Harvard Medical School, Boston, MA 02115.
↵ ‖ Present address: Defense Intelligence Agency, Arlington VA, 22201.
↵ †† To whom reprint requests should be addressed at: National Cancer Institute, Laboratory of Cellular and Molecular Biology, Building 37, Room 1E24, 37 Convent Drive, Medical Science Center 4255, Bethesda, MD 20892-4255. e-mail: rubinj{at}dc37a.nci.nih.gov.
Harold E. Varmus
Data deposition: The sequence in this paper has been deposited in the GenBank database (accession no. AF001900).
ABBREVIATIONS:

FRP,

Frizzled-related protein;

FZ,

Frizzled;

CRD,

cysteine-rich domain of Frizzled proteins;

HGF/SF,

hepatocyte growth factor/scatter factor