Susan Cumberledge

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 domain homologous to the putative Wnt-binding site of Frizzleds. To facilitate the biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields ∼3 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.

The Drosophila Wnt, Wingless, Provides an Essential Signal for Pre- and Postsynaptic Differentiation

At vertebrate neuromuscular junctions (NMJs), Agrin plays pivotal roles in synapse development, but molecules that activate synapse formation at central synapses are largely unknown. Members of the Wnt family are well established as morphogens, yet recently they have also been implicated in synapse maturation. Here we demonstrate that the Drosophila Wnt, Wingless (Wg), is essential for synapse development. We show that Wg and its receptor are expressed at glutamatergic NMJs, and that Wg is secreted by synaptic boutons. Loss of Wg leads to dramatic reductions in target-dependent synapse formation, and new boutons either fail to develop active zones and postsynaptic specializations or these are strikingly aberrant. We suggest that Wg signals the coordinated development of pre- and postsynaptic compartments.

Syndecan-1 is required for Wnt-1-induced mammary tumorigenesis in mice

Syndecan-1 is a cell-surface, heparan-sulphate proteoglycan (HSPG) predominantly expressed by epithelial cells. It binds specifically to many proteins, including oncoproteins. For example, it induces the assembly of a signalling complex between FGF ligands and their cognate receptors. But so far there has been no direct evidence that this proteoglycan contributes to tumorigenesis. Here we have examined the role of syndecan-1 (encoded by Sdc1) during mammary tumour formation in response to the ectopic expression of the proto-oncogene Wnt1. We crossed syndecan-1–deficient mice with transgenic mice that express Wnt1 in mammary gland (TgN(Wnt-1)1Hev; ref. 2). Ectopic Wnt-1 expression induces generalized mammary hyperplasia, followed by the development of solitary tumours (median time 22 weeks). We show that in Sdc1−/− mice, Wnt-1–induced hyperplasia in virgin mammary gland was reduced by 70%, indicating that the Wnt-1 signalling pathway was inhibited. Of the 39 tumours that developed in a test cohort of mice, only 1 evolved in the Sdc1−/− background. In addition, we show that soluble syndecan-1 ectodomain purified from mouse mammary epithelial cells stimulates the activity of a Wnt-1 homologue in a tissue culture assay. Our results provide both genetic and biochemical evidence that syndecan-1 can modulate Wnt signalling, and is critical for Wnt-1–induced tumorigenesis of the mouse mammary gland.

Wingless signaling at synapses is through cleavage and nuclear import of receptor DFrizzled2.

Wingless secretion provides pivotal signals during development by activating transcription of target genes. At Drosophila synapses, Wingless is secreted from presynaptic terminals and is required for synaptic growth and differentiation. Wingless binds the seven-pass transmembrane DFrizzled2 receptor, but the ensuing events at synapses are not known. We show that DFrizzled2 is endocytosed from the postsynaptic membrane and transported to the nucleus. The C terminus of DFrizzled2 is cleaved and translocated into the nucleus; the N-terminal region remains just outside the nucleus. Translocation of DFrizzled2-C into the nucleus, but not its cleavage and transport, depends on Wingless signaling. We conclude that, at synapses, Wingless signal transduction occurs through the nuclear localization of DFrizzled2-C for potential transcriptional regulation of synapse development.

Glycosaminoglycans and WNTs: just a spoonful of sugar helps the signal go down

New studies have brought Drosophila geneticists face to face with some perplexing questions about proteoglycans and their role in growth factor signaling. Three papers published in Development report that mutations in the gene encoding UDP-glucose dehydrogenase disrupt both the synthesis of glycosaminoglycans and WINGLESS/WNT signaling 1-3, while a fourth paper provides evidence that TGF-~/DPP signaling Js regulated by the proteoglycan DALLY (Ref. 4).

Sequence homology between Wingless/Wnt-1 and a lipid-binding domain in secreted phospholipase A2.

We have found that the carboxyl terminus of Wingless/Wnt-1 shares significant sequence homology with a lipid-binding domain found in class I and class II secreted phospholipase A2 (sPLA2) proteins. The Drosophila Wingless protein and its vertebrate ortholog, Wnt-1, belong to the Wnt family of secreted growth factors [1]. Wnt activity is modulated by a number of extracellular factors, including cell-surface proteoglycans [2] and two types of secreted antagonists, the Frizzled-related proteins [3] and Dickkopf-1 (Dkk-1) [4]. Lipids have been implicated in Wnt signaling before, when Aravind and Koonin [5] hypothesized that Dkk-1 inhibition of Wnt signaling involves membrane localization. Dkk-1 has been linked to lipids because of the presence of a putative colipase fold in the Dkks [5]. The colipase proteins assist pancreatic lipases in lipid digestion. When pancreatic lipases are unable to bind directly to a lipid interface (for example, in the presence of bile salts), colipases function as co-factors, promoting close contact between the lipase and the lipid interface. X-ray crystallographic studies [6,7] of the colipase–lipase complex have identified several conserved hydrophobic residues on the colipase that contact the lipid interface. The putative colipase fold present in the Dkks includes some of these conserved hydrophobic residues [5]. If Dkks and colipases share functional as well as structural homology, then Dkks might be expected to promote lipid–protein interactions. The amino acid sequence similarity between the carboxyl terminus of Wingless/Wnt-1 and the lipid-binding region of sPLA2s further implicates membrane interactions in Wnt signaling. The sPLA2 enzymes hydrolyze the headgroup of specific phospholipids at the sn-2 position [8]. Four classes of sPLA2s have been categorized and the class I and II sPLA2s are highly conserved [9,10]. Sequence database analysis using the BLASTP [11,12] and FASTA [13] programs detected a 50 amino acid region of the class I and II sPLA2s that is 30–40% identical to the highly conserved carboxyterminal domain of Wingless/Wnt-1. Figure 1 shows the conservation between the two protein families, as assessed using the ‘strong homology’ class of the Gonnet-PAM250 mutation data matrix [14]. The snake (Agkistrodon piscivorus piscivorus) class II sPLA2 (App-K49) and Wingless are 37% identical and 44% conserved in this 50 amino acid region, while the bovine pancreatic class I sPLA2 and Wingless are 28% identical and 35% conserved. This level of homology extends to other Wnt-1 proteins (Figure 1) and is similar among all members of the Wnt family. Within the 50 amino acid region, important structural features have been conserved between the two families. All catalytically active PLA2s contain two ‘signature’ consensus sequences [15] that include active-site residues. One of these signature sequences, CCXXHXXC (in the single-letter amino acid code, where X is any amino acid), is present within the 50 amino acid region (Figure 1, Magazine R353

A lipid-binding domain in Wnt: a case of mistaken identity?

The alignments of sPLA2s and Wnts of Reichsman et al. [4xSequence homology between Wingless/Wnt-1 and a lipid-binding domain in secreted phospholipase A2. Reichsman, F, Moore, HM, and Cumberledge, S. Curr Biol. 1999; 9: R353–R355Abstract | Full Text | Full Text PDF | PubMedSee all References[4] and Barnes and Russell [17xA lipid-binding domain in Wnt: a case of mistaken identity?. Barnes, MR and Russell, RB. Curr Biol. 1999; 9: R717–R718Abstract | Full Text | Full Text PDF | PubMed | Scopus (1)See all References[17] were generated using two different paradigms. Barnes and Russell compared the physico-chemical properties of the amino acid residues; we used the Gonnet PAM 250 substitution matrix to evaluate amino acid similarities. PAM (percentage of acceptable point mutations) matrices have been widely recommended for detecting related proteins [[18]xSchwartz, RM and Dayhoff, MO. : 353–362See all References, [19]xThe significance of protein sequence similarities. Collins, JF, Coulson, AFW, and Lyall, A. Comp App Biosci. 1988; 4: 67–71PubMedSee all References, [20]xAmino acid substitution matrices from an information theoretic perspective. Altschul, S. J Mol Biol. 1991; 219: 555–565Crossref | PubMed | Scopus (389)See all References]. These substitution matrices are based on evolutionary relationships between proteins and are generated by analyzing point mutations in closely related proteins. Using the PAM matrices, we find that sPLA2 App-K49 residues 9–61 are more similar to the carboxy-terminal portion of Wingless than to the corresponding bovine sPLA2 sequences (44% versus 36% similarity). A total of 24 residues are conserved between sPLA2 and Wingless; 7 of these are cysteines. Note that App-K49 lacks the conserved Ala49, does not bind Ca2+ and has no catalytic activity, yet retains the ability to bind membranes and disrupt phospholipid vesicles [21xThe growing phospholipase A2 superfamily of signal transduction enzymes. Dennis, EA. Trends Biochem Sci. 1997; 22: 1–2Abstract | Full Text PDF | PubMed | Scopus (705)See all References[21].The first 60 residues of sPLA2 form the helix–loop–helix backbone that constitutes much of the lipid-binding pocket. The amino-terminal helix and loop are important for interfacial membrane binding, whereas residues in helix 2 contribute to the active site. As the rest of the App-K49 sequence has no similarity to Wingless, the two protein families probably have very different tertiary structures. Site-directed mutagenesis studies on sPLA2 [22xPhospholipase A2 engineering. The roles of disulfide bonds in structure, conformational stability, and catalytic function. Zho, H, Dupureur, CM, Zhang, X, and Tsai, MD. Biochemistry. 1995; 34: 15307–15314Crossref | PubMedSee all References[22] have shown that of the seven conserved disulphide bonds, only one, Cys29–Cys45, is required for lipase activity. Cys29 and Cys45 correspond to Cys428 and Cys445 in Wingless. These cysteines are conserved in the Wnts, and Cys445 is required for Wingless activity. Nonetheless, we agree that it is difficult to evaluate the significance of these sequence similarities without more information on Wnt function and structure.