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• W2067836746 abstract "Mammalian Vangl1 and Vangl2 are highly conserved membrane proteins that have evolved from a single ancestral protein Strabismus/Van Gogh found in Drosophila. Mutations in the Vangl2 gene cause a neural tube defect (craniorachischisis) characteristic of the looptail (Lp) mouse. Studies in model organisms indicate that Vangl proteins play a key developmental role in establishing planar cell polarity (PCP) and in regulating convergent extension (CE) movements during embryogenesis. The role of Vangl1 in these processes is virtually unknown, and the molecular function of Vangl1 and Vangl2 in PCP and CE is poorly understood. Using a yeast two-hybrid system, glutathione S-transferase pull-down and co-immunoprecipitation assays, we show that both mouse Vangl1 and Vangl2 physically interact with the three members of the cytoplasmic Dishevelled (Dvl) protein family. This interaction is shown to require both the predicted cytoplasmic C-terminal half of Vangl1/2 and a portion of the Dvl protein containing PDZ and DIX domains. In addition, we show that the two known Vangl2 loss-of-function mutations identified in two independent Lp alleles associated with neural tube defects impair binding to Dvl1, Dvl2, and Dvl3. These findings suggest a molecular mechanism for the neural tube defect seen in Lp mice. Our observations indicate that Vangl1 biochemical properties parallel those of Vangl2 and that Vangl1 might, therefore, participate in PCP and CE either in concert with Vangl2 or independently of Vangl2 in discrete cell types. Mammalian Vangl1 and Vangl2 are highly conserved membrane proteins that have evolved from a single ancestral protein Strabismus/Van Gogh found in Drosophila. Mutations in the Vangl2 gene cause a neural tube defect (craniorachischisis) characteristic of the looptail (Lp) mouse. Studies in model organisms indicate that Vangl proteins play a key developmental role in establishing planar cell polarity (PCP) and in regulating convergent extension (CE) movements during embryogenesis. The role of Vangl1 in these processes is virtually unknown, and the molecular function of Vangl1 and Vangl2 in PCP and CE is poorly understood. Using a yeast two-hybrid system, glutathione S-transferase pull-down and co-immunoprecipitation assays, we show that both mouse Vangl1 and Vangl2 physically interact with the three members of the cytoplasmic Dishevelled (Dvl) protein family. This interaction is shown to require both the predicted cytoplasmic C-terminal half of Vangl1/2 and a portion of the Dvl protein containing PDZ and DIX domains. In addition, we show that the two known Vangl2 loss-of-function mutations identified in two independent Lp alleles associated with neural tube defects impair binding to Dvl1, Dvl2, and Dvl3. These findings suggest a molecular mechanism for the neural tube defect seen in Lp mice. Our observations indicate that Vangl1 biochemical properties parallel those of Vangl2 and that Vangl1 might, therefore, participate in PCP and CE either in concert with Vangl2 or independently of Vangl2 in discrete cell types. Neural tube closure is a complex developmental process that takes place early during embryogenesis and is a key step in formation of the central nervous system, including spinal cord (1Copp A.J. Greene N.D.E. Murdoch J.N. Nat. Rev. Genet. 2003; 4: 784-793Crossref PubMed Scopus (542) Google Scholar). In humans, failure of the neural tube to close properly results in a group of syndromes collectively known as neural tube defects (NTDs), 1The abbreviations used are: NTD, neural tube defect; TM, transmembrane; PCP, planar cell polarity; RT, reverse transcription; aa, amino acid(s); CE, convergent extension; CMV, cytomegalovirus; DBD, DNA binding domain; AD, activation domain; SD, synthetic medium; GST, glutathione S-transferase. which constitute the second most frequent cause of congenital abnormalities (1 in 1000 live births) (2Frey L. Hauser W.A. Epilepsia. 2003; 44: 4-13Crossref PubMed Scopus (225) Google Scholar). The cellular and molecular mechanisms underlying NTDs are very complex, poorly understood, and difficult to study in humans. Recent genetic studies of NTDs in model organisms have identified a number of genes and proteins that play critical roles in neural tube closure (1Copp A.J. Greene N.D.E. Murdoch J.N. Nat. Rev. Genet. 2003; 4: 784-793Crossref PubMed Scopus (542) Google Scholar). In our laboratory, we have identified Vangl2 (Van Gogh-like, formerly “loop-tail associated protein,” Ltap) as the gene mutated in the mouse model of severe NTD known as looptail (Lp) (3Kibar Z. Vogan K.J. Groulx N. Justice M.J. Underhill D.A. Gros P. Nat. Genet. 2001; 28: 251-255Crossref PubMed Scopus (395) Google Scholar, 4Kibar Z. Underhill D.A. Canonne-Hergaux F. Gauthier S. Justice M.J. Gros P. Genomics. 2001; 72: 331-337Crossref PubMed Scopus (40) Google Scholar). Heterozygous Lp animals (Lp/+) are normal except for the presence of a characteristic “kinked” (looped) tail, whereas Lp homozygotes (Lp/Lp) die at mid-gestation due to craniorachischisis, a very severe NTD that is characterized by a completely open neural tube from the midbrain/hindbrain boundary to the caudal (tail) region (3Kibar Z. Vogan K.J. Groulx N. Justice M.J. Underhill D.A. Gros P. Nat. Genet. 2001; 28: 251-255Crossref PubMed Scopus (395) Google Scholar, 4Kibar Z. Underhill D.A. Canonne-Hergaux F. Gauthier S. Justice M.J. Gros P. Genomics. 2001; 72: 331-337Crossref PubMed Scopus (40) Google Scholar, 5Murdoch J.N. Doudney K. Patemotte C. Copp A.J. Stanier P. Hum. Mol. Genet. 2001; 10: 2593-2601Crossref PubMed Scopus (270) Google Scholar). Two alleles have been described for the Lp mice: naturally occurring Lp (3Kibar Z. Vogan K.J. Groulx N. Justice M.J. Underhill D.A. Gros P. Nat. Genet. 2001; 28: 251-255Crossref PubMed Scopus (395) Google Scholar) and a chemically induced Lpm1Jus (4Kibar Z. Underhill D.A. Canonne-Hergaux F. Gauthier S. Justice M.J. Gros P. Genomics. 2001; 72: 331-337Crossref PubMed Scopus (40) Google Scholar). Vangl2 is a 521-amino acid transmembrane (TM) protein composed of four putative TM domains in the N-terminal half. The C-terminal half is predicted to be cytoplasmic and is possibly involved in intracellular signaling and/or interaction with other proteins (3Kibar Z. Vogan K.J. Groulx N. Justice M.J. Underhill D.A. Gros P. Nat. Genet. 2001; 28: 251-255Crossref PubMed Scopus (395) Google Scholar, 4Kibar Z. Underhill D.A. Canonne-Hergaux F. Gauthier S. Justice M.J. Gros P. Genomics. 2001; 72: 331-337Crossref PubMed Scopus (40) Google Scholar, 5Murdoch J.N. Doudney K. Patemotte C. Copp A.J. Stanier P. Hum. Mol. Genet. 2001; 10: 2593-2601Crossref PubMed Scopus (270) Google Scholar, 6Park M. Moon R.T. Nat. Cell Biol. 2002; 4: 20-25Crossref PubMed Scopus (309) Google Scholar). Vangl2 mRNA is embryonically expressed in a number of tissues, including the neural tube immediately prior to, during, and after closure (3Kibar Z. Vogan K.J. Groulx N. Justice M.J. Underhill D.A. Gros P. Nat. Genet. 2001; 28: 251-255Crossref PubMed Scopus (395) Google Scholar, 4Kibar Z. Underhill D.A. Canonne-Hergaux F. Gauthier S. Justice M.J. Gros P. Genomics. 2001; 72: 331-337Crossref PubMed Scopus (40) Google Scholar, 5Murdoch J.N. Doudney K. Patemotte C. Copp A.J. Stanier P. Hum. Mol. Genet. 2001; 10: 2593-2601Crossref PubMed Scopus (270) Google Scholar). The NTD phenotype of both Lp mutants (Lp, Lpm1Jus) is associated with independent missense mutations within the Vangl2 cytoplasmic domain affecting amino acid residues otherwise conserved in the protein family: S464N (Lp) and D255E (Lpm1Jus) (3Kibar Z. Vogan K.J. Groulx N. Justice M.J. Underhill D.A. Gros P. Nat. Genet. 2001; 28: 251-255Crossref PubMed Scopus (395) Google Scholar, 4Kibar Z. Underhill D.A. Canonne-Hergaux F. Gauthier S. Justice M.J. Gros P. Genomics. 2001; 72: 331-337Crossref PubMed Scopus (40) Google Scholar). The similar phenotypic consequences of heterozygosity (-/+) and homozygosity (-/-) at independent Lp alleles (4Kibar Z. Underhill D.A. Canonne-Hergaux F. Gauthier S. Justice M.J. Gros P. Genomics. 2001; 72: 331-337Crossref PubMed Scopus (40) Google Scholar) and the strict recessive mode of inheritance of Lp-associated NTD strongly suggest that Lp mutations behave as loss-of-function mutations in a gene dosage-sensitive pathway, signifying a role for Vangl2 as a critical regulator of neural tube formation. Vangl2 orthologs have been found in flies (Drosophila), worms (Caenorhabditis elegans), zebrafish (Danio rerio), and mammals, thus defining a family of evolutionarily conserved proteins (7Katoh M. Intl. J. Mol. Med. 2002; 10: 11-15PubMed Google Scholar). Interestingly, analysis of fish, mouse, and human genomes has identified two Vangl homologous genes in each species designated Vangl1 and Vangl2. Originally cloned in Drosophila by two groups, the primordial gene was given the dual appellation Strabismus (Stbm) or Van Gogh (Vang), based on the phenotypic appearance of certain structures in fly mutants (8Taylor J. Abramova N. Charlton J. Adler P.N. Genetics. 1998; 150: 199-210PubMed Google Scholar, 9Wolff T. Rubin G.M. Development. 1998; 125: 1149-1159PubMed Google Scholar). The gene was shown to play a role in establishing planar cell polarity (PCP), or the orientation of epithelial cells in a planar layer (10Gubb D. Garcia-Bellido A. J. Embryol. Exp. Morphol. 1982; 68: 37-57PubMed Google Scholar, 11Strutt D. Development. 2003; 130: 4501-4513Crossref PubMed Scopus (213) Google Scholar). In flies, disruption of the PCP pathway through inactivation of one of several so-called “core PCP genes” results in mis-orientation of normally highly organized specific epithelial structures such as ommatidia of eye, hairs on the wing cells, bristles on the legs, and others (11Strutt D. Development. 2003; 130: 4501-4513Crossref PubMed Scopus (213) Google Scholar, 12Mlodzik M. Trends Genet. 2002; 18: 564-571Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). In addition to Stbm/Vang, other core PCP proteins include the seven TM domain receptor Frizzled (Fz), an atypical cadherin Starry night/Flamingo (Stan/Fml), and two cytoplasmic proteins, Dishevelled (Dsh/Dvl) and Prickle (Pk) (13Klingensmith J. Nusse R. Perrimon N. Genes Dev. 1994; 8: 118-130Crossref PubMed Scopus (344) Google Scholar, 14Theisen H. Purcell J. Bennett M. Kansagara D. Syed A. Marsh J.L. Development. 1994; 120: 347-360Crossref PubMed Google Scholar, 15Chae J. Kim M.J. Goo J.H. Collier S. Gubb D. Charlton J. Adler P.N. Park W.J. Development. 1999; 126: 5421-5429PubMed Google Scholar, 16Gubb D. Green C. Huen D. Coulson D. Johnson G. Tree D. Collier S. Roote J. Genes Dev. 1999; 13: 2315-2327Crossref PubMed Scopus (223) Google Scholar, 17Usui T. Shima Y. Shimada Y. Hirano S. Burgess R.W. Schwarz T.L. Takeichi M. Uemura T. Cell. 1999; 98: 585-595Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar). Extensive studies in flies have shown that the establishment of proper planar polarity relies on the formation of a multiprotein membrane complex consisting of core PCP proteins Stbm/Vang, Pk, Dvl, and Fz. In individual cells of the fly wing, PCP proteins are initially arranged symmetrically at the apical membrane, but become asymmetrically redistributed during the establishment of PCP. A complex formed by Stbm/Vang and Pk assumes an apical-proximal localization, whereas a Fz-Dvl complex is redirected to the apical-distal portion of the cell (18Axelrod J.D. Genes Dev. 2001; 15: 1182-1187PubMed Google Scholar, 19Tree D.R.P. Shulman J.M. Rousset R. Scott M.P. Gubb D. Axelrod J.D. Cell. 2002; 109: 371-381Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 20Bastock R. Strutt H. Strutt D. Development. 2003; 130: 3007-3014Crossref PubMed Scopus (251) Google Scholar). This asymmetry is believed to be key in establishing planar polarity (11Strutt D. Development. 2003; 130: 4501-4513Crossref PubMed Scopus (213) Google Scholar). At the molecular level, fly Stbm and its vertebrate orthologVangl2 have been shown to interact directly with cytoplasmic Dvl and Pk proteins leading to their recruitment to the membrane (6Park M. Moon R.T. Nat. Cell Biol. 2002; 4: 20-25Crossref PubMed Scopus (309) Google Scholar, 20Bastock R. Strutt H. Strutt D. Development. 2003; 130: 3007-3014Crossref PubMed Scopus (251) Google Scholar, 21Takeuchi M. Nakabayashi J. Sakaguchi T. Yamamoto T.S. Takahashi H. Takeda H. Ueno N. Curr. Biol. 2003; 13: 674-679Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 22Carreira-Barbosa F. Concha M.L. Takeuchi M. Ueno N. Wilson S.W. Tada M. Development. 2003; 130: 4037-4046Crossref PubMed Scopus (213) Google Scholar, 23Jenny A. Darken R.S. Wilson P.A. Mlodzik M. EMBO J. 2003; 22: 4409-4420Crossref PubMed Scopus (177) Google Scholar). Although the function of Pk in PCP is not well understood, the role of Dvl has been better characterized. Dvl proteins have a modular organization typical of adaptor proteins with three structurally conserved domains, the N-terminal DIX domain, the central PDZ domain, and the C-terminal DEP domain (24Boutros M. Paricio N. Strutt D.I. Mlodzik M. Cell. 1998; 94: 109-118Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, 25Boutros M. Mlodzik M. Mech. Dev. 1999; 83: 27-37Crossref PubMed Scopus (235) Google Scholar, 26Moriguchi T. Kawachi K. Kamakura S. Masuyama N. Yamanaka H. Matsumoto K. Kikuchi A. Nishida E. J. Biol. Chem. 1999; 274: 30957-30962Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Dvl has been demonstrated to play key roles in both canonical Wnt and PCP pathways (reviewed in Ref. 27Veeman M.T. Axelrod J.D. Moon R.T. Dev. Cell. 2003; 5: 367-377Abstract Full Text Full Text PDF PubMed Scopus (1163) Google Scholar). The DIX domain is indispensable for canonical Wnt signaling, whereas the DEP domain is dedicated to the PCP pathway and the PDZ functions in both (25Boutros M. Mlodzik M. Mech. Dev. 1999; 83: 27-37Crossref PubMed Scopus (235) Google Scholar, 29Penton A. Wodarz A. Nusse R. Genetics. 2002; 161: 747-762Crossref PubMed Google Scholar). 2R. Nusse (2001). The Wnt gene homepage, www.stanford.edu/~rnusse/wntwindow.html. Mutations in the DEP domain interfere with Dvl translocation to the membrane (18Axelrod J.D. Genes Dev. 2001; 15: 1182-1187PubMed Google Scholar, 30Axelrod J.D. Miller J.R. Shulman J.M. Moon R.T. Perrimon N. Genes Dev. 1998; 12: 2610-2622Crossref PubMed Scopus (543) Google Scholar). Recent studies in vertebrates have revealed that Vangl2 regulates the process of convergent extension (CE, controlled by the non-canonical Wnt pathway) during gastrulation and neurulation in frogs and zebrafish (6Park M. Moon R.T. Nat. Cell Biol. 2002; 4: 20-25Crossref PubMed Scopus (309) Google Scholar, 31Goto T. Keller R. Dev. Biol. 2002; 247: 165-181Crossref PubMed Scopus (187) Google Scholar, 32Darken R.S. Scola A.M. Rakeman A.S. Das G. Mlodzik M. Wilson P.A. EMBO J. 2002; 21: 976-985Crossref PubMed Scopus (196) Google Scholar, 33Jessen J.R. Topczewski J. Bingham S. Sepich D.S. Marlow F. Chandrasekhar A. Somica-Krezel L. Nat. Cell Biol. 2002; 4: 610-615Crossref PubMed Scopus (403) Google Scholar). During CE, dorsal mesenchymal and neuroepithelial cells become polarized in a medio-lateral direction, migrate, and intercalate. These directional cellular activities result in concomitant lengthening of the anterior-posterior axis and narrowing of the epithelial mass in a perpendicular direction (34Keller R. Science. 2002; 298: 1950-1954Crossref PubMed Scopus (572) Google Scholar). Failure of CE in mesenchyme produces animals with shorter, wider trunks (34Keller R. Science. 2002; 298: 1950-1954Crossref PubMed Scopus (572) Google Scholar, 35Wallingford J.B. Rowning B.A. Vogeli K.M. Rothbacher U. Fraser S.E. Harland R.M. Nature. 2000; 405: 81-85Crossref PubMed Scopus (635) Google Scholar), whereas CE distortion during neural tube formation leads to significant widening of the neural plate in the midline and, as a result, the neural tube folds are spaced too far apart to permit closure (1Copp A.J. Greene N.D.E. Murdoch J.N. Nat. Rev. Genet. 2003; 4: 784-793Crossref PubMed Scopus (542) Google Scholar, 36Wallingford J.B. Harland R.M. Development. 2002; 129: 5815-5825Crossref PubMed Scopus (266) Google Scholar). The phenotype of the Lp mouse is compatible with manifestations of CE failure, because the body axis is shortened, the neural plate is broadened, and the neural tube does not close (3Kibar Z. Vogan K.J. Groulx N. Justice M.J. Underhill D.A. Gros P. Nat. Genet. 2001; 28: 251-255Crossref PubMed Scopus (395) Google Scholar, 4Kibar Z. Underhill D.A. Canonne-Hergaux F. Gauthier S. Justice M.J. Gros P. Genomics. 2001; 72: 331-337Crossref PubMed Scopus (40) Google Scholar, 5Murdoch J.N. Doudney K. Patemotte C. Copp A.J. Stanier P. Hum. Mol. Genet. 2001; 10: 2593-2601Crossref PubMed Scopus (270) Google Scholar). Interestingly, mutations in mammalian counterparts of the Drosophila PCP proteins Flamingo (Celsr1) (37Curtin J.A. Quint E. Tsipouri V. Arkell R.M. Cattanach B. Copp A.J. Henderson D.J. Steel N. Brown S.D.M. Gray I.C. Murdoch J.N. Curr. Biol. 2003; 13: 1129-1133Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar) or Dishevelled (Dvl1;Dvl2 double knockout) (38Hamblet N.S. Lijam N. Ruiz-Lozano P. Wang J. Yang Y. Luo Z. Mei L. Chien K.R. Sussman D.J. Wynshaw-Boris A. Development. 2002; 129: 5827-5838Crossref PubMed Scopus (381) Google Scholar) cause phenotypes indistinguishable from that of the Lp mouse. These observations have prompted the hypothesis that the regulation of CE in vertebrates is related to the PCP pathway in flies, including a critical role for vertebrate relatives of PCP proteins such as Stbm/Vang, Dvl, Fz, and Pk (reviewed in Ref. 39Fanto M. McNeill H. J. Cell Sci. 2004; 117: 527-533Crossref PubMed Scopus (186) Google Scholar). The physiological role and biochemical properties of Vangl2 are now being unraveled, yet Vangl1 function remains a mystery. In the present study, we aimed to characterize the mammalian Vangl1 protein. To determine whether structural similarities in the Vangl family translate into conservation of function we investigated possible interactions of Vangl1 and Vangl2 with three known members of the Dvl family. We also examined the effect of Lp mutations of Vangl2 on Vangl2 interactions with Dvl proteins and demonstrated that the S464N and D255E mutations dramatically decrease Vangl2 binding to Dvl proteins. These findings suggest a molecular explanation for the loss-of-function of Vangl2 protein in Lp mouse and establish a critical role for Vangl2:Dvl interaction in proper signaling during neural tube closure. Plasmids—Total RNA from mouse embryos (E14.5) was used as a template for reverse transcriptase and polymerase chain reaction amplification (RT-PCR) of cDNAs corresponding to mouse Vangl1 and Vangl2 with Taq-HiFi Polymerase (Invitrogen), as previously described (3Kibar Z. Vogan K.J. Groulx N. Justice M.J. Underhill D.A. Gros P. Nat. Genet. 2001; 28: 251-255Crossref PubMed Scopus (395) Google Scholar). cDNA products corresponding to the proposed cytoplasmic domains of mouse Vangl1 (251–526 aa) and Vangl2 (238–521 aa) were subcloned into plasmid vector pBDT7 (Clontech) to yield pBD-mVangl1 and pBD-mVangl2 and into plasmid pGEX4T (Amersham Biosciences) to produce pGST-mVangl1 and pGST-mVangl2. Restriction enzyme sites (in brackets and underlined in sequences) were incorporated within oligonucleotide primers for in-frame insertion of Vangl1/2 cDNAs in corresponding fusion proteins. The following oligonucleotide primers were used: pBD-mVangl1: 5′-CAAAGAGAATTCATGTTCACCCTGCAGGTGGTC-3′ (EcoRI) and 5′-TTCTCTGGATCCCTTAGACTGATGTCTCAGACTG-3′ (BamHI); pBD-mVangl2: 5′-CAAGAGCATATGGAGCTCCGTCAGCTCCAGCCC-3′ (NdeI) and 5′-GTTCTCGAATTCCTGCTGCAAAAGTCACACAGA-3′ (EcoRI); pGST-mVangl1: 5′-CAAGAGGGATCCATGTTCACCCTGCAGGTGGTC-3′ (BamHI) and 5′-TTCTCTGAATTCTTAGACTGATGTCTCAGACTG-3′ (EcoRI); pGST-mVangl2: 5′-CAAGAGGAATTCGAGCTCCGTCAGCTCCAGCCC-3′ (EcoRI); and 5′-TTCCTCGTCGACCCTGCAAAAGTCACACAGA-3′ (SalI). Full-length cDNAs for mouse Vangl1 and Vangl2 were generated and cloned into pCB6 mammalian expression vector. A short antigenic epitope (EQKLISEEDL) derived from the human c-Myc protein (c-Myc tag) was inserted in-frame at the N terminus of Vangl1 and in the predicted extracytoplasmic domain of Vangl2 (following amino acid position 136). The c-Myc epitope sequence is indicated in bold. The following oligonucleotide primer pairs were used: pCMV-Vangl1: 5′-CAAGAAGGTACCATTGCTATGGAGCAGAAGCTAATCTCTGAGGAGGATCTGGATACCGAATCCACG-3′ (KpnI) and 5′-CTCTTGAAGCTTTTAGACTGATGTCTCAGACTG-3′ (HindIII); pCMV-Vangl2: fragment A 5′-CATAGCGGTACCATGGACACCGAGTCCCAGTAC-3′ (KpnI); 5′-CAGATCCTCCTCAGAGATTAGCTTCTGCTCGGCCGTCCCACACGGCTCCTGCTC-3; and fragment B 5′-GAGCAGAAGCTAATCTCTGAGGAGGATCTGGAGCTGGAGCCGT GTGGGACG-3′ and 5′-CTCTTGAAGCTTAAGTCACACAGAGGTCTC-3′ (HindIII). Two separate Vangl2 fragments A and B were initially generated by RT-PCR. The obtained PCR products were gel-purified, mixed in 1:1 proportion, incubated at 95 °C for 10 min, and slowly cooled down to 30 °C. Final amplification was done with the forward primer of segment A and the reverse primer of segment B. Fragments of the three mouse Dishevelled genes, mDvl1, mDvl2, and mDvl3, were generated by RT-PCR and subcloned into plasmid vector pGADT7 (Stratagene). These included portions corresponding to (a) the N-terminal halves (1–404 aa, 1–418 aa, and 1–395 aa) of the Dvl1/2/3 proteins, respectively, (b) the C-terminal portion of Dvl3 (389–717 aa), and (c) the predicted DIX domain (1–87 aa) of Dvl1. The following oligonucleotide primers were used: pAD-mDvl1–5′: 5′-GAAAGAGAATTCARGGCGGAGACCAAAATCATCTACCACATGGACGAG-3′ (EcoRI) and 5′-CTCTTGATCGATGCTACGGCGCCTCCTCAAGCTGTGG-3′ (ClaI); pAD-mDvl2–5′: 5′-CATAGCATCGATGGATGGCGGGCAGCAGCGCGGGG-3′ (ClaI) and 5′-GATACGGGATCCCAGAGAGACCCCGGCCTTCGCA-3′ (BamHI); pAD-mDvl3–5′: 5′-CATAGCGAATTCATGGGCGAGACCAAGATCATCTAC-3′ (EcoRI) and 5′-ATCAGTATCGATCGATGGAGCTGGTGATGGAGGA-3′ (ClaI); pAD-mDvl3–3′: 5′-CATAGCGAATTCATCACCAGCTCCATC-3′ (EcoRI) and 5′-ATCAGTATCGATCGGGCCCTGATCACATCACATC-3′ (ClaI); pAD-mDvl1-DIX: 5′-GAAAGAGAATTCARGGCGGAGACCAAAATCATCTACCACATGGAC GAG-3′ (EcoRI) and 5′-CATCAAATCGATAGCGCCCTCAGCCAGGACCAGCCA-3′ (ClaI). Amplification products were digested with indicated enzymes, gel-purified using a QIAEX II gel extraction kit (Qiagen), and subcloned into yeast expression plasmid (see “Results”). The sequence integrity of all PCR-amplified inserts was verified by sequencing. Full-length cDNA clones for mouse Dvl1 (tagged with a c-Myc epitope), Dvl2 and Dvl3 (tagged with an HA epitope) engineered into CMV-driven mammalian expression vector were kindly provided by Dr. X. Li (Department of Genetics and Developmental Biology, University of Connecticut Health Center). Yeast Two-hybrid System—The commercially available yeast two-hybrid system MatchMaker system 3 (#K1612–1, Clontech) was used to study possible interactions between Vangl1/Vangl2 proteins and different domains of Dishevelled. The procedures used for these studies were exactly as described by the manufacturer (Clontech). The system is based on the use of Saccharomyces cerevisiae strain AH109, which is engineered to have three different reporters, namely ADE2, HIS3, and lacZ (cytoplasmic β-galactosidase and secreted α-galactosidase) under the control of distinct GAL4 upstream regulatory sequences derived from the GAL2, GAL1, and MEL1 gene promoters, respectively. These three reporters yield strong and specific response to GAL4, and the simultaneous use of ADE2 and HIS3 as selectable markers eliminates false positives. The system also includes two plasmid vectors: pGBKT7 (Kanr, TRP1, and GAL4DBD c-Myc-tagged) and pGADT7 (Ampr, LEU2, and GAL4AD HA-tagged) that direct the production of recombinant proteins consisting of independent fusions to the DNA binding domain (DBD) or activation domain (AD) of GAL4. The two test plasmids are introduced by transformation into either AH109 (MATa) or Y187 (MATα), both strains auxotrophs for leucine, tryptophan, histidine, and adenine. Individual pGBKT7 and pGADT7 constructs were transformed into yeast cells using a lithium acetate transformation procedure, and expression of the corresponding recombinant GAL4 proteins was confirmed by immunoblotting of whole cell lysates with either anti-c-Myc (9E10, BAbCO) or anti-HA (16B12, BAbCO) mouse monoclonal antibodies. Possible reconstituted GAL4 activity was tested in diploids obtained by mating AH109 and Y187 transformants positive for expression of the test GAL4 fusions followed by plating on synthetic medium (SD) of various stringencies: low stringency lacking tryptophan and leucine (SD-Trp/-Leu), medium stringency lacking histidine, tryptophan, and leucine (SD-His/-Trp/-Leu), and high stringency lacking adenine, histidine, tryptophan, and leucine (SD-Ade/-His/-Trp/-Leu). Substrate 5-bromo-4-chloro-3-indolyl-α-d-galactopyranoside (X-α-gal) was used to visualize LacZ-producing clones directly on the plates. Protein-protein interactions were evaluated by assessing growth of diploid cells in solid medium after 72 h at 30 °C. Cell Lines and Transfections—To study possible interactions of Vangl1/2 proteins with Dvls in mammalian cells, pCB6-Vangl1, pCB6-Vangl2, and pCMV-Dvl1/2/3 (human cytomegalovirus promotor/enhancer) were introduced by transient transfection into human embryonic kidney 293 (HEK293) cells using the commercial reagent LipofectAMINE 2000 (Invitrogen). Control pCB6 plasmid DNA was used to adjust the total amount of transfected DNA to 4 μg/plate. Twenty-four hours following transfection, cells were washed with cold phosphate-buffered saline, scraped, and lysed in PLC buffer containing 50 mm Hepes (pH 7.5), 150 mm NaCl, 10% glycerol, 0.5% Triton X-100, 1.5 mm MgC2, 1 mm EGTA, protease inhibitors phenylmethylsulfonyl fluoride (1 mm), leupeptin and aprotinin (each at 10 μg/ml), and sodium vanadate (200 μm). These lysates were prepared on ice and stored frozen or used immediately for co-immunoprecipitation and GST pull-down experiments. Co-immunoprecipitation and GST Pull-down Assays—For co-immunoprecipitation experiments, lysates from transiently transfected HEK293 cells (500 μl) were incubated with either 1 μg of appropriate mouse monoclonal antibodies Dvl1 (3F12), Dvl2 (10B5), Dvl3 (4D3) (Santa Cruz Biotechnology), or with 1:250 dilution of rabbit polyclonal anti-Vangl1 and anti-Vangl2 antisera (see below) together with 25 μl of a slurry of Protein A/G coupled to agarose beads (Invitrogen) for 16 h at 4 °C on a rotating wheel. Protein A/G beads were washed twice by centrifugation at 4 °C in a cold buffer consisting of 20 mm HEPES (pH 7.5), 500 mm NaCl, 10% glycerol, 0.5% Triton X-100, 1.5 mm MgCl2, 10 mm NaPO4, pH 7.5, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml of leupeptin and aprotinin, followed by two additional washes in the same buffer containing 200 mm NaCl. Immune complexes were eluted by incubation of the protein A/G beads in 2× sample buffer (2% SDS, 2 m β2-mercaptoethanol) at 100 °C, 10 min. Proteins were separated by electrophoresis on SDS-containing acrylamide gels (SDS-PAGE) followed by immunoblotting with polyclonal anti-Vangl1 and anti-Vangl2 antibodies. A portion of each cell lysate (1/20) was resolved on PAGE-SDS and immunoblotted with anti-Dvl or anti-Vangl1/2 antibodies to control for input loading. For GST pull-down assays, GST-Vangl1, GST-Vangl2, and GST (from empty pGEX4T) fusion proteins were purified from Escherichia coli Bl21cells. Cell growth, induction conditions, cell lysis, and isolation of GST fusions by affinity chromatography were exactly as described by the manufacturer of the pGEX4T plasmid (Amersham Biosciences). GST-fused proteins were stored on glutathione-Sepharose 4B beads (Amersham Biosciences) at -80 °C. Equal amounts of GST-Vangl1, GST-Vangl2, and control GST proteins on beads (with respect to protein concentration) were mixed with either recombinant Dvl proteins produced by transient transfection in HEK293 cells (see above) or with Dvl proteins present in cell lysates from E13.5 mouse embryos prepared with PLC buffer. Mixtures were rotated at 4 °C for 4–16 h, washed five times with cold 50 mm Tris, pH7.4, 150 mm NaCl, 0.5% Triton X-100, 1.5 mm MgCl2, and 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml of each leupeptin and aprotinin, and eluted with 2× Laemmli sample buffer. Proteins were resolved by SDS-PAGE and analyzed by immunoblotting using anti-mouse monoclonal antibodies Dvl1 (3F12), Dvl2 (10B5), Dvl3 (4D3), and GST (B-14) (Santa Cruz Biotechnology). Antibody Production—Vangl1 (14–70 aa) and Vangl2 (12–64 aa) polypeptides fused to glutathione S-transferase (GST) were used as immunogens to raise polyclonal rabbit sera in male New Zealand White rabbits, as described previously (40Vidal S.M. Pinner E. Lepage P. Gauthier S. Gros P. J. Immunol. 1996; 157: 3559-3568PubMed Google Scholar). The Vangl1 and Vangl2 polypeptides were cloned into the pQE-40 (Qiagen) expression vector for production of Vangl1/2 fusion proteins containing six consecutive histidines and a portion of dihydrofolate reductase (6-His-DFHR) to be used for purification of anti-Vangl1 and anti-Vangl2 antibodies by affinity chromatography, as we described (41Gruenheid S. Canonne-Hergaux F. Gauthier S. Hackam D.J. Grinstein S. Gros P. J. Exp. Med. 1999; 189: 831-841Crossref PubMed Scopus (267) Google Scholar). Vangl1 and Vangl2 polypeptides were amplified by RT-PCR using the following primers: pGST-Vangl1ab: 5′-CAAGAAGGATCCTACTCAAGCCATTCCAAAAAATC-3′ (BamH1) and 5′-CAAGAGGAATTCCTGAACTTCCTCTGCGCCTGT-3′ (EcoR1); pGST-Vangl2ab: 5′-CAAGAAGGATCCTATTCCTACAAGTCGG (BamH1) and 5′-CAAGAGGAATTCCCTCGTGGACTCATTG-3′ (EcoR1); pDFHR-Vangl1: 5′-CAAGAAGGTACCTACTCAAGCCATTCCAAAAAATC-3′ (KpnI) and 5′-CAAGAGCTGCAGCTGAACTTCCTCTGCGCCTGT-3′ (PstI); pDFHR-Vangl2: 5′-CAAGAAGGTACCTATTCCT" @default.
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• W2067836746 date "2004-12-01" @default.
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• W2067836746 title "Independent Mutations in Mouse Vangl2 That Cause Neural Tube Defects in Looptail Mice Impair Interaction with Members of the Dishevelled Family" @default.
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• W2067836746 doi "https://doi.org/10.1074/jbc.m408675200" @default.
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