Marcel Wehrli

arrow encodes an LDL-receptor-related protein essential for Wingless signalling.

The Wnt family of secreted molecules functions in cell-fate determination and morphogenesis during development in both vertebrates and invertebrates (reviewed in ref. 1). Drosophila Wingless is a founding member of this family, and many components of its signal transduction cascade have been identified, including the Frizzled class of receptor. But the mechanism by which the Wingless signal is received and transduced across the membrane is not completely understood. Here we describe a gene that is necessary for all Wingless signalling events in Drosophila. We show that arrow gene function is essential in cells receiving Wingless input and that it acts upstream of Dishevelled. arrow encodes a single-pass transmembrane protein, indicating that it may be part of a receptor complex with Frizzled class proteins. Arrow is a low-density lipoprotein (LDL)-receptor-related protein (LRP), strikingly homologous to murine and human LRP5 and LRP6. Thus, our data suggests a new and conserved function for this LRP subfamily in Wingless/Wnt signal reception.

Wg/Wnt Signal Can Be Transmitted through Arrow/LRP5,6 and Axin Independently of Zw3/Gsk3β Activity

Activation of the Wnt signaling cascade provides key signals during development and in disease. Here we provide evidence, by designing a Wnt receptor with ligand-independent signaling activity, that physical proximity of Arrow (LRP) to the Wnt receptor Frizzled-2 triggers the intracellular signaling cascade. We have uncovered a branch of the Wnt pathway in which Armadillo activity is regulated concomitantly with the levels of Axin protein. The intracellular pathway bypasses Gsk3beta/Zw3, the kinase normally required for controlling beta-catenin/Armadillo levels, suggesting that modulated degradation of Armadillo is not required for Wnt signaling. We propose that Arrow (LRP) recruits Axin to the membrane, and that this interaction leads to Axin degradation. As a consequence, Armadillo is no longer bound by Axin, resulting in nuclear signaling by Armadillo.

Cloning and characterization of αPS1, a novel Drosophila melanogaster integrin

Abstract The Drosophila position-specific integrins (PS integrins or PS antigens) comprise two heterodimeric complexes, αPS1βPS and αPS2βPS. With the cloning of αPS1 described here, we complete the characterization of the primary structure of the three PS integrin subunits. We have purified the αPS1 subunit, obtained peptide sequence and isolated genomic and cDNA clones. The encoded αPS1 protein contains pattern of the cleaved alpha integrins, three putative metal binding domains and shows the other characteristic features of alpha integrins. Regions of sequence variation indicate that αPS1 is distinct from all other alpha chains. The transcript analysis shows that the patterns of both αPS1 mRNA and protein expression are the same, suggesting that the gene is controlled transcriptionally. We compare the gene structures of the DrosophilaαPS1, αPS2, the human αPS1and αPS2 (p150,95) and the C. elegans F54G8.3 integrins. We find several positions and phases of introns conserved which, supported by conservation also in the amino acid sequence, indicates that they all derive from a common ancestral gene.

Unexpectedly robust assembly of the Axin destruction complex regulates Wnt/Wg signaling in Drosophila as revealed by analysis in vivo

Secreted proteins in the Wnt family regulate gene expression in target cells by causing the accumulation of the transcriptional activator beta-catenin. In the absence of Wnt, a protein complex assembled around the scaffold protein Axin targets beta-catenin for destruction, thereby preventing it from transducing inappropriate signals. Loss of Axin or its binding partners APC and GSK3 results in aberrant activation of the Wnt signaling response. We have analyzed the effects of mutant forms of Drosophila Axin with large internal deletions when expressed at physiological levels in vivo, either in the presence or absence of wild type Axin. Surprisingly, even deletions that completely remove the binding sites for fly APC, GSK3 or beta-catenin, though they fail to rescue to viability, these mutant forms of Axin cause only mild developmental defects, indicating largely retained Axin function. Furthermore, two lethal Axin deletion constructs, AxinDeltaRGS and AxinDeltabeta cat(DeltaArm), can complement each other and restore viability. Our findings support a model in which the Axin complex is assembled through cooperative tripartite interactions among the binding partners, making the assembly of functional complexes surprisingly robust.

Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 sites of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range.

correction: arrow encodes an LDL-receptor-related protein essential for Wingless signalling

This corrects the article DOI: 35035110

In vivo analysis in Drosophila reveals differential requirements of contact residues in Axin for interactions with GSK3β or β-catenin

Proper regulation of the Wingless/Wnt signaling pathway is essential for normal development. The scaffolding protein Axin plays a key role in this process through interactions with Drosophila Shaggy and Armadillo. In the current studies, we used a yeast two-hybrid assay to identify ten amino acids in Axin that are critical for in vitro interaction with Shaggy and two for interaction with Armadillo. We then generated five Axin variants in which individual putative contact amino acids were mutated and compared their activity, as assayed by rescue of axin null mutant flies, to that of Axin lacking the entire Shaggy (AxinDeltaSgg) or Armadillo (AxinDeltaArm) binding domain. Although we expected these mutants to function identically to Axin in which the entire binding domain was deleted, we instead observed a spectrum of phenotypic rescue. Specifically, two point mutants within the Shaggy binding domain showed loss of activity similar to that of AxinDeltaSgg and dominantly interfered with complex function, whereas a third mutant allele, AxinK446E, retained most function. Two Axin point mutants within the Armadillo binding domain were weak alleles and retained most function. These findings demonstrate the importance of in vivo verification of the role of specific amino acids within a protein.