title: Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes.
abstract: BACKGROUND
  Mutations in the Planar Cell Polarity (PCP) core gene Vangl2 cause the most severe neural tube defects (NTD) in mice and humans. Genetic studies show that the Vangl2 gene genetically interacts with a close homologue Vangl1. How precisely Vangl2 and Vangl1 proteins interact and crosstalk has remained a difficult issue to address, with the main obstacle being the accurate discrimination of the two proteins, which share close sequence homology. Experimental evidence previously presented has been sparse and addressed with ectopically expressed proteins or with antibodies unable to biochemically discriminate Vangl1 from Vangl2, therefore giving rise to unclear results.
  
  
  RESULTS
  A highly specific monoclonal anti-Vangl2 antibody was generated and rigorously tested on both recombinant and extracted Vangl2 using surface plasmon resonance (SPR) analysis, western blot, and immunoprecipitation experiments. This antibody efficiently affinity-purified Vangl2 from cell lysates and allowed the unambiguous identification of endogenous Vangl2 by proteomic analysis. Vangl1 was also present in Vangl2 immunoprecipitates, establishing the first biochemical evidence for the existence of Vangl2/Vangl1 heterodimers at an endogenous level. Epitope-tagged Vangl2 and Vangl1 confirmed that both proteins interact and colocalize at the plasma membrane. The Vangl2 antibody is able to acutely assess differential expression levels of Vangl2 protein in culture cell lines, as corroborated with gene expression analysis. We characterised Vangl2 expression in the cochlea of homozygous and heterozygous Lp mutant mice bearing a point mutation within the C-terminal Vangl2 region that leads to profound PCP defects. Our antibody could detect much lower levels of Vangl2(Lp) protein in mutant mice compared to the wild type mice.
  
  
  CONCLUSIONS
  Our results provide an in-depth biochemical characterisation of the interaction observed between Vangl paralogues.
authors.affiliation: Inserm U1068, CRCM, Marseille, France.
authors.forename: Edwige
authors.initials: E
authors.lastname: Belotti
authors.forename: Tania M
authors.initials: TM
authors.lastname: Puvirajesinghe
authors.forename: Stéphane
authors.initials: S
authors.lastname: Audebert
authors.forename: Emilie
authors.initials: E
authors.lastname: Baudelet
authors.forename: Luc
authors.initials: L
authors.lastname: Camoin
authors.forename: Michel
authors.initials: M
authors.lastname: Pierres
authors.forename: Lea
authors.initials: L
authors.lastname: Lasvaux
authors.forename: Géraldine
authors.initials: G
authors.lastname: Ferracci
authors.forename: Mireille
authors.initials: M
authors.lastname: Montcouquiol
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Borg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D016764:Cell Polarity / Q000235:genetics; D015870:Gene Expression; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009436:Neural Tube Defects / Q000175:diagnosis / Q000235:genetics*; D017354:Point Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D040901:Proteomics; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; C431577:Ltap protein, mouse; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C528120:Vangl1 protein, mouse