A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors - PubMed (original) (raw)

A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors

A Rattner et al. Proc Natl Acad Sci U S A. 1997.

Abstract

This paper describes the identification of a new family of mammalian genes that encode secreted proteins containing homology to the cysteine-rich ligand-binding domain found in the frizzled family of transmembrane receptors. The secreted frizzled-related proteins (sFRPs) are approximately 30 kDa in size, and each contains a putative signal sequence, a frizzled-like cysteine-rich domain, and a conserved hydrophilic carboxy-terminal domain. The sFRPs are not the products of differential splicing of the known frizzled genes. Glycosylphosphatidylinositol-anchored derivatives of sFRP-2 and sFRP-3 produced in transfected human embryonic kidney cells confer cell-surface binding by the Drosophila Wingless protein. These observations suggest that sFRPs may function in vivo to modulate Wnt signaling, or, alternatively, as novel ligands for as yet unidentified receptors.

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Figures

Figure 1

(Left) Alignment of mouse sFRP amino acid sequences. The alignment includes the CRD of Drosophila frizzled (3), indicated by fz(CRD). The sFRP-4 sequence is incomplete; it is derived from a segment of genomic DNA that encompasses a single exon of the sFRP-4 gene. (Right) Dendrogram of CRD sequences in which the length of each horizontal line is proportional to the degree of amino acid sequence divergence. The region used to construct the dendrogram extends from the 1st to the 10th cysteine of the CRD. smo, smoothened; coll XVIII, collagen XVIII. Species origins are indicated by an uppercase letter: C, C. elegans; D, Drosophila; H, human; M, mouse; R, rat.

Figure 1

Figure 2

Tissue distribution of sFRP transcripts in the adult mouse and rat. (Left) Ten micrograms of total mouse RNA from the indicated tissues was used for RNase protection with each of the sFRP probes. A control reaction with a RNA polymerase II probe is shown at the bottom. The sFRP-4 RNase protection autoradiogram was exposed three times longer than the other autoradiograms. (Right) Twenty micrograms of total rat RNA from the indicated tissues was used for Northern blot hybridization with each of the sFRP probes. A control probe derived from ribosomal protein S26 is shown at the bottom of the sFRP-3 Northern blot. The sFRP-4 probe failed to produce a detectable signal under the hybridization conditions used. The mobilities of 18S and 28S ribosomal RNAs are indicated to the right. B, brain; E, eye; H, heart; K, kidney; Li, liver; Lu, lung; R, retina; S, spleen; T, testis; Y, yeast tRNA.

Figure 3

In situ localization of sFRP-1 transcripts in the adult mouse brain and eye. 33P in situ hybridization is shown in red, superimposed upon a cresyl violet stain shown in black and white. (A) In the brain, the hybridization signal is seen in the two lateral ventricles and in the central third ventricle. (B) In the eye, the hybridization signal is seen in the anterior epithelium of the lens and in the ciliary body. The cornea is facing to the left in both sections.

Figure 4

Partial chromosome linkage maps showing the locations of the sFRP-1, sFRP-2, sFRP-3, and sFRP-4 genes in the mouse. The genes were mapped by interspecific backcross analysis. To the left of each chromosome map, the number of recombinant N2 animals divided by the total number of N2 animals typed for each pair of loci is presented. The recombination frequencies, expressed as genetic distance in centimorgans (± one standard error) are also shown. The upper 95% confidence limit of the recombination distance is given in parentheses when no recombinants were found between loci. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns. The positions of loci on human chromosomes, where known, are shown to the right of the chromosome maps. References for the map positions of most human loci can be obtained from the Genome Data Base, a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).

Figure 5

sFRP-2 and sFRP-3 bind to Drosophila Wingless when they are expressed as GPI-anchored cell-surface proteins. 293 cells were transiently transfected either with sFRP-2 (A and B) or with sFRP-3 (C and D) expression constructs in which the sFRP open reading frames were linked at their carboxy termini to a myc epitope-tagged GPI anchor sequence. The cells were incubated with conditioned medium containing Wingless protein, immunostained with affinity purified anti-Wingless antibodies, and examined by confocal microscopy. (A and C) Immunofluorescent staining. (B and D) Phase contrast.

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A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors - PubMed (original) (raw)