Molecular Characterisation of Endogenous Vangl2/Vangl1 Heteromeric Protein Complexes
PLoS ONE. 2012-09-28; 7(9): e46213
DOI: 10.1371/journal.pone.0046213
Read on PubMed
1. PLoS One. 2012;7(9):e46213. doi: 10.1371/journal.pone.0046213. Epub 2012 Sep 28.
Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein
complexes.
Belotti E(1), Puvirajesinghe TM, Audebert S, Baudelet E, Camoin L, Pierres M,
Lasvaux L, Ferracci G, Montcouquiol M, Borg JP.
Author information:
(1)Inserm U1068, CRCM, Marseille, France.
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.
METHODOLOGY AND MAIN FINDINGS: 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.
CONCLUSION: Our results provide an in-depth biochemical characterisation of the
interaction observed between Vangl paralogues.
DOI: 10.1371/journal.pone.0046213
PMCID: PMC3460870
PMID: 23029439 [Indexed for MEDLINE]