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Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes.

Authors :
Edwige Belotti
Tania M Puvirajesinghe
Stéphane Audebert
Emilie Baudelet
Luc Camoin
Michel Pierres
Lea Lasvaux
Géraldine Ferracci
Mireille Montcouquiol
Jean-Paul Borg
Source :
PLoS ONE, Vol 7, Iss 9, p e46213 (2012)
Publication Year :
2012
Publisher :
Public Library of Science (PLoS), 2012.

Abstract

BackgroundMutations 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.Methodology and main findingsA 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.ConclusionOur results provide an in-depth biochemical characterisation of the interaction observed between Vangl paralogues.

Subjects

Subjects :
Medicine
Science

Details

Language :
English
ISSN :
19326203
Volume :
7
Issue :
9
Database :
Directory of Open Access Journals
Journal :
PLoS ONE
Publication Type :
Academic Journal
Accession number :
edsdoj.87709adb080a4559b31b0d6be284f9a3
Document Type :
article
Full Text :
https://doi.org/10.1371/journal.pone.0046213