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, 94 (13), 6770-5

Purification and Molecular Cloning of a Secreted, Frizzled-related Antagonist of Wnt Action

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Purification and Molecular Cloning of a Secreted, Frizzled-related Antagonist of Wnt Action

P W Finch et al. Proc Natl Acad Sci U S A.

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 approximately 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.

Figures

Figure 1
Figure 1
(A) SDS/PAGE analysis of heparin-Sepharose purified FRP. Approximately 200 ng of protein was resolved in a 4–20% polyacrylamide minigel (Novex) under reducing (+) or nonreducing (−) conditions, and subsequently stained with silver. The position of molecular mass markers is indicated at the right. (B) Representation of human FRP cDNA clones. Overlapping clones HS1 and HS8 are shown above a diagram of the complete coding sequence and the adjacent 5′ and 3′ untranslated regions. The coding region is boxed; the open portion corresponds to the signal sequence. Untranslated regions are represented by a line. Selected restriction sites are indicated. (C) Predicted FRP amino acid sequence (standard single-letter code). The peptide sequence obtained from the purified protein is underlined. Double-underlined sequences were used to generate oligonucleotide probes for screening of the M426 cDNA library. The putative signal sequence is italicized. The large shaded region is the cysteine-rich domain homologous to CRDs in members of the FZ family. The small shaded region is a lysine-rich segment that fulfills the criteria for a consensus hyaluronic acid-binding sequence. The dashed underlining denotes two potential asparagine-linked glycosylation sites.
Figure 2
Figure 2
Comparison of the CRDs of FRP and other members of the FZ family. Solid black shading highlights identities present in human FRP and any other FZ family member. The consensus sequence indicates residues present in at least 8 of the 16 FZ or FZ-related proteins. ∗∗, The ten invariant cysteine residues; single asterisks indicate other invariant residues. hFRP, human FZ-related protein; hFZ (36); hFZ5 (16); mFZ3-mFZ8 (16); rFZ1 and rFZ2 (37); dFZ (21); dFZ2 (17); cFZ (16); mCOL, mouse collagen XVIII (38); hFRZB (39); and mSDF5 (40).
Figure 3
Figure 3
FRP mRNA expression in normal human adult and embryonic tissues, and in cultured cells. Blots containing ≈2 μg of poly(A)+ RNA from each of the indicated tissues or 10 μg of total RNA from different human cell lines were probed with radiolabeled FRP and β-actin cDNA fragments, as described in the Methods. The position of DNA size markers, expressed in kb, is indicated at the left of the tissue blots; the position of 28S and 18S ribosomal RNA is shown at the left of the cell line blot.
Figure 4
Figure 4
Chromosomal localization of the FRP gene by fluorescent in situ hybridization. To localize the FRP gene, 100 sets of metaphase chromosomes were analyzed. In 80 metaphases, a double fluorescent signal was observed with the FRP genomic probe in 8p11.1-12 on both chromosome homologs (Left). The identity of the chromosomes was confirmed by hybridization with a probe specific for chromosome 8 (Right).
Figure 5
Figure 5
Southern blot analysis of FRP genomic sequences in different species. After fractionation by agarose gel electrophoresis and transfer to filters, EcoRI-digested genomic DNAs were hybridized in the presence of either 50% or 35% formamide. Specimens were from the following species: λ, lamda phage; H, human; Mk, rhesus monkey; Mo, mouse; C, chicken; X, Xenopus laevis; D, Drosophila melanogaster; Y, yeast (S. cerevisiae).
Figure 6
Figure 6
Biosynthesis of FRP in M426 cells. A pulse-chase experiment was performed with metabolically labeled cells incubated either in the absence or presence of heparin. Proteins were immunoprecipitated from cell lysates (C.L.) or conditioned medium (C.M.) with FRP peptide antiserum in the absence or presence of competing peptide, and resolved in a SDS/10% polyacrylamide gel. Cells and media were harvested 1, 4, or 20 h after a 30 min labeling period. Lanes 1–24 are labeled at the bottom. The protein band corresponding to FRP is indicated by an arrow. The position of molecular mass markers is shown at the left.
Figure 7
Figure 7
Dorsal axis duplication in Xenopus embryos in response to varying combinations of Wnt and FRP transcripts. Each bar represents the percentage of axis duplication; the solid portion within each bar represents the percentage of extensive duplication, which is defined by the presence of the cement gland and at least one eye in the duplicated axis. The total number of embryos injected in two to four independent experiments is indicated by the number above each bar. The amount of mRNA injected per embryo is shown below the bars.

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