Rap1 regulation of RIAM and cell adhesion

Methods Enzymol. 2006;407:345-58. doi: 10.1016/S0076-6879(05)07029-1.

Abstract

The small GTPase Rap1 has been involved in different cellular processes. Rap1 is known to increase cell adhesion by means of integrin activation, to induce cell spreading, and to regulate adherent junctions at cell-cell contacts. How Rap1 mediates these cell responses is poorly known, but currently developing evidence points to the involvement of different effector pathways. Recently, we described RIAM, a Rap1 interacting adaptor protein that regulates integrin activation and hence cell adhesion. RIAM is required for Rap1-induced adhesion and seems to control Rap1 localization at the plasma membrane, where Rap1 regulates integrin activation. In this chapter, we focus in the role of RIAM in regulating Rap1-mediated cell adhesion. We describe the method for studying the Rap1-RIAM interaction using in vitro and in vivo approaches such as yeast two hybrids, pull-down assays. and coimmunoprecipitation. The role of Rap1 and RIAM in integrin-mediated adhesion is studied by cell adhesion assays to immobilized integrin substrates and by changes in integrin activation as determined by activation epitope exposure. Finally, we describe an approach to determine the role of RIAM in regulating intracellular localization of active Rap1.

MeSH terms

  • Adaptor Proteins, Signal Transducing / physiology
  • Cell Adhesion / drug effects
  • Cell Adhesion / physiology*
  • Escherichia coli / metabolism
  • Fibronectins / metabolism
  • Humans
  • Integrins / metabolism
  • Intercellular Adhesion Molecule-1 / metabolism
  • Jurkat Cells
  • Membrane Proteins / physiology
  • Protein Interaction Mapping
  • Transfection
  • Two-Hybrid System Techniques
  • rap1 GTP-Binding Proteins / isolation & purification
  • rap1 GTP-Binding Proteins / physiology*

Substances

  • APBB1IP protein, human
  • Adaptor Proteins, Signal Transducing
  • Fibronectins
  • Integrins
  • Membrane Proteins
  • Intercellular Adhesion Molecule-1
  • rap1 GTP-Binding Proteins