The role of the cysteine‐rich domain of Frizzled in Wingless‐Armadillo signaling

The Frizzled (Fz) receptors contain seven transmembrane helices and an amino‐terminal cysteine‐rich domain (CRD) that is sufficient and necessary for binding of the ligands, the Wnts. Recent genetic experiments have suggested, however, that the CRD is dispensable for signaling. We engineered fz CRD mutant transgenes and tested them for Wg signaling activity. None of the mutants was functional in cell culture or could fully replace fz in vivo. We also show that replacing the CRD with a structurally distinct Wnt‐binding domain, the Wnt inhibitory factor, reconstitutes a functional Wg receptor. We therefore hypothesized that the function of the CRD is to bring Wg in close proximity with the membrane portion of the receptor. We tested this model by substituting Wg itself for the CRD, a manipulation that results in a constitutively active receptor. We propose that Fz activates signaling in two steps: Fz uses its CRD to capture Wg, and once bound Wg interacts with the membrane portion of the receptor to initiate signaling.

[1]  Stephen M. Mount,et al.  The genome sequence of Drosophila melanogaster. , 2000, Science.

[2]  P. Ingham,et al.  smoothened encodes a receptor-like serpentine protein required for hedgehog signalling , 1996, Nature.

[3]  T. Bouwmeester,et al.  Signaling specificity by Frizzled receptors in Drosophila. , 2000, Science.

[4]  Randall T Moon,et al.  A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. , 2003, Developmental cell.

[5]  Nobuyuki Onishi,et al.  The receptor tyrosine kinase Ror2 is involved in non‐canonical Wnt5a/JNK signalling pathway , 2003, Genes to cells : devoted to molecular & cellular mechanisms.

[6]  R. Nusse,et al.  Wingless Repression of Drosophila frizzled 2 Expression Shapes the Wingless Morphogen Gradient in the Wing , 1998, Cell.

[7]  Andrew Tomlinson,et al.  arrow encodes an LDL-receptor-related protein essential for Wingless signalling , 2000, Nature.

[8]  N. Perrimon,et al.  wingless refines its own expression domain on the Drosophila wing margin , 1996, Nature.

[9]  R. Nusse,et al.  Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless. , 2000, Molecular cell.

[10]  R. Nusse,et al.  The Wnt signaling pathway in development and disease. , 2004, Annual review of cell and developmental biology.

[11]  V. Katanaev,et al.  Trimeric G Protein-Dependent Frizzled Signaling in Drosophila , 2005, Cell.

[12]  J. Nathans,et al.  Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[13]  R. Nusse,et al.  Ligand Receptor Interactions in the Wnt Signaling Pathway inDrosophila * , 2002, The Journal of Biological Chemistry.

[14]  R. J. Hill,et al.  C. elegans LIN-18 Is a Ryk Ortholog and Functions in Parallel to LIN-17/Frizzled in Wnt Signaling , 2004, Cell.

[15]  R. Nusse,et al.  Genetic Evidence That Drosophila frizzled Controls Planar Cell Polarity and Armadillo Signaling by a Common Mechanism , 2005, Genetics.

[16]  R. Nusse,et al.  WntD is a feedback inhibitor of Dorsal/NF-κB in Drosophila development and immunity , 2005, Nature.

[17]  R. Carthew,et al.  Use of dsRNA-Mediated Genetic Interference to Demonstrate that frizzled and frizzled 2 Act in the Wingless Pathway , 1998, Cell.

[18]  I. Weissman,et al.  Wnt proteins are lipid-modified and can act as stem cell growth factors , 2003, Nature.

[19]  W. Strapps,et al.  Transducing properties of Drosophila Frizzled proteins. , 2001, Development.

[20]  A. Penzo-Méndez,et al.  The C‐terminal cytoplasmic Lys‐Thr‐X‐X‐X‐Trp motif in frizzled receptors mediates Wnt/β‐catenin signalling , 2000, The EMBO journal.

[21]  Ajamete Kaykas,et al.  Zebrafish Prickle, a Modulator of Noncanonical Wnt/Fz Signaling, Regulates Gastrulation Movements , 2003, Current Biology.

[22]  M. Mlodzik,et al.  Subcellular Localization of Frizzled Receptors, Mediated by Their Cytoplasmic Tails, Regulates Signaling Pathway Specificity , 2004, PLoS biology.

[23]  Yoichi Kato,et al.  LDL-receptor-related proteins in Wnt signal transduction , 2000, Nature.

[24]  N. Perrimon,et al.  Zygotic lethal mutations with maternal effect phenotypes in Drosophila melanogaster. II. Loci on the second and third chromosomes identified by P-element-induced mutations. , 1996, Genetics.

[25]  Eric Wieschaus,et al.  Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity. , 2003, Developmental cell.

[26]  C. M. Chen,et al.  Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila. , 1999, Development.

[27]  Feng Cong,et al.  Wnt signals across the plasma membrane to activate the β-catenin pathway by forming oligomers containing its receptors, Frizzled and LRP , 2004, Development.

[28]  N. Perrimon,et al.  Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. , 1993, Development.

[29]  P. Adler,et al.  Molecular analysis of EMS-induced frizzled mutations in Drosophila melanogaster. , 1996, Genetics.

[30]  Jeffrey D. Axelrod,et al.  A Second Canon , 2003 .

[31]  A. Bejsovec.  Wnt Pathway Activation New Relations and Locations , 2005, Cell.

[32]  P. Adler,et al.  Directional non-cell autonomy and the transmission of polarity information by the frizzled gene of Drosophila , 1987, Nature.

[33]  H. Varmus,et al.  Wnt/Wingless signaling through β-catenin requires the function of both LRP/Arrow and frizzled classes of receptors , 2003, BMC Cell Biology.

[34]  Eric Wieschaus,et al.  Rethinking WNT signaling. , 2004, Trends in genetics : TIG.

[35]  M. Noll,et al.  The Drosophila smoothened Gene Encodes a Seven-Pass Membrane Protein, a Putative Receptor for the Hedgehog Signal , 1996, Cell.

[36]  P. Adler,et al.  A single frizzled protein has a dual function in tissue polarity. , 1994, Development.

[37]  Michael J Parker,et al.  Mutations in LRP5 or FZD4 underlie the common familial exudative vitreoretinopathy locus on chromosome 11q. , 2004, American journal of human genetics.

[38]  J. Nathans,et al.  A new secreted protein that binds to Wnt proteins and inhibits their activites , 1999, Nature.

[39]  J. Zhang,et al.  Interactions between Wingless and DFz2 during Drosophila wing development. , 1998, Development.

[40]  J. Nathans,et al.  Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development. , 1999, Development.

[41]  Wange Lu,et al.  Mammalian Ryk Is a Wnt Coreceptor Required for Stimulation of Neurite Outgrowth , 2004, Cell.

[42]  John B. Thomas,et al.  Wnt-mediated axon guidance via the Drosophila Derailed receptor , 2003, Nature.

[43]  G. Garriga,et al.  The Caenorhabditis elegans Ror RTK CAM-1 Inhibits EGL-20/Wnt Signaling in Cell Migration , 2004, Genetics.

[44]  Jeremy Nathans,et al.  A new member of the frizzled family from Drosophila functions as a Wingless receptor , 1996, Nature.

[45]  Y. Ip,et al.  Drosophila WntD is a target and an inhibitor of the Dorsal/Twist/Snail network in the gastrulating embryo , 2005, Development.

[46]  G. Struhl,et al.  Evidence that the cysteine-rich domain of Drosophila Frizzled family receptors is dispensable for transducing Wingless. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[47]  J. Nathans,et al.  Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains , 2001, Nature.

[48]  K. Ui-Tei,et al.  Dfrizzled-3, a new Drosophila Wnt receptor, acting as an attenuator of Wingless signaling in wingless hypomorphic mutants. , 1999, Development.