The structure of Rap1 in complex with RIAM reveals specificity determinants and recruitment mechanism

J Mol Cell Biol. 2014 Apr;6(2):128-39. doi: 10.1093/jmcb/mjt044. Epub 2013 Nov 28.

Abstract

The small GTPase Rap1 induces integrin activation via an inside-out signaling pathway mediated by the Rap1-interacting adaptor molecule (RIAM). Blocking this pathway may suppress tumor metastasis and other diseases that are related to hyperactive integrins. However, the molecular basis for the specific recognition of RIAM by Rap1 remains largely unknown. Herein we present the crystal structure of an active, GTP-bound GTPase domain of Rap1 in complex with the Ras association (RA)-pleckstrin homology (PH) structural module of RIAM at 1.65 Å. The structure reveals that the recognition of RIAM by Rap1 is governed by side-chain interactions. Several side chains are critical in determining specificity of this recognition, particularly the Lys31 residue in Rap1 that is oppositely charged compared with the Glu31/Asp31 residue in other Ras GTPases. Lys31 forms a salt bridge with RIAM residue Glu212, making it the key specificity determinant of the interaction. We also show that disruption of these interactions results in reduction of Rap1:RIAM association, leading to a loss of co-clustering and cell adhesion. Our findings elucidate the molecular mechanism by which RIAM mediates Rap1-induced integrin activation. The crystal structure also offers new insight into the structural basis for the specific recruitment of RA-PH module-containing effector proteins by their small GTPase partners.

Keywords: RA–PH; RIAM; Rap1; crystal structure; inside-out signaling; integrin signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry*
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Amino Acid Sequence
  • Amino Acids / metabolism
  • Animals
  • Cell Adhesion
  • Cell Membrane / metabolism
  • Crystallography, X-Ray
  • Humans
  • Integrins / metabolism
  • Membrane Proteins / chemistry*
  • Membrane Proteins / metabolism*
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Protein Binding
  • Protein Structure, Tertiary
  • Reproducibility of Results
  • Structural Homology, Protein
  • Substrate Specificity
  • Telomere-Binding Proteins / chemistry*
  • Telomere-Binding Proteins / metabolism*
  • ras Proteins / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • Amino Acids
  • Integrins
  • Membrane Proteins
  • RIAM protein, mouse
  • TERF2IP protein, human
  • Telomere-Binding Proteins
  • ras Proteins