Analysis of the interaction of Plexin-B1 and Plexin-B2 with Rnd family proteins

Thomas Wylie, Ritu Garg, Anne J Ridley, Maria R Conte

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Abstract

The Rnd family of proteins, Rnd1, Rnd2 and Rnd3, are atypical Rho family GTPases, which bind to but do not hydrolyse GTP. They interact with plexins, which are receptors for semaphorins, and are hypothesised to regulate plexin signalling. We recently showed that each Rnd protein has a distinct profile of interaction with three plexins, Plexin-B1, Plexin-B2 and Plexin-B3, in mammalian cells, although it is unclear which region(s) of these plexins contribute to this specificity. Here we characterise the binary interactions of the Rnd proteins with the Rho-binding domain (RBD) of Plexin-B1 and Plexin-B2 using biophysical approaches. Isothermal titration calorimetry (ITC) experiments for each of the Rnd proteins with Plexin-B1-RBD and Plexin-B2-RBD showed similar association constants for all six interactions, although Rnd1 displayed a small preference for Plexin-B1-RBD and Rnd3 for Plexin-B2-RBD. Furthermore, mutagenic analysis of Rnd3 suggested similarities in its interaction with both Plexin-B1-RBD and Plexin-B2-RBD. These results suggest that Rnd proteins do not have a clear-cut specificity for different Plexin-B-RBDs, possibly implying the contribution of additional regions of Plexin-B proteins in conferring functional substrate selection.

Original languageEnglish
Pages (from-to)e0185899
JournalPLoS ONE
Volume12
Issue number10
Early online date17 Oct 2017
DOIs
Publication statusPublished - 17 Oct 2017

Keywords

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • COS Cells
  • Calorimetry
  • Cercopithecus aethiops
  • Cloning, Molecular
  • Escherichia coli
  • Gene Expression
  • Kinetics
  • Mutation
  • Nerve Tissue Proteins
  • Neural Cell Adhesion Molecules
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Receptors, Cell Surface
  • Recombinant Proteins
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Thermodynamics
  • rho GTP-Binding Proteins
  • Journal Article

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