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Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin
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Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

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Binding of the cytoplasmic domain of intercellular adhesion molecule-2 (ICAM-2) to alpha-actinin

L Heiska1, C Kantor, T Parr

  • 1Department of Pathology, Division of Biochemistry, University of Helsinki, FIN-00014 Helsinki, Finland.

The Journal of Biological Chemistry
|October 18, 1996
PubMed

Insights

Intercellular adhesion molecule-2 (ICAM-2) interacts with alpha-actinin, a key cytoskeletal protein. This binding links cell surface adhesion molecules to the actin cytoskeleton, crucial for leukocyte adhesion.

Area of Science:

  • Cell Biology
  • Immunology
  • Molecular Biology

Background:

  • Intercellular adhesion molecule-2 (ICAM-2) is vital for leukocyte adhesion, acting as a ligand for lymphocyte function-associated antigen-1 (LFA-1).
  • Understanding ICAM-2's intracellular interactions is crucial for elucidating leukocyte trafficking and immune responses.

Purpose of the Study:

  • To investigate the intracellular binding partners of ICAM-2.
  • To identify the specific regions of ICAM-2 and alpha-actinin involved in their interaction.
  • To clarify the role of alpha-actinin in linking ICAM-2 to the cytoskeleton.

Main Methods:

  • Affinity matrix assay using a peptide of ICAM-2's cytoplasmic domain (amino acids 231-254).
  • Immunoblotting to identify bound proteins, specifically alpha-actinin.
  • Confocal microscopy to visualize ICAM-2 and alpha-actinin colocalization in Eahy926 cells.
  • Peptide competition assays and analysis of alpha-actinin fusion proteins to map interaction sites.

Main Results:

  • Alpha-actinin was identified as a binding protein for the ICAM-2 cytoplasmic peptide.
  • Purified alpha-actinin directly bound to the ICAM-2 peptide.
  • Confocal microscopy confirmed colocalization of ICAM-2 and alpha-actinin in cells.
  • The ICAM-2(241-248) octapeptide showed strong binding to alpha-actinin, and multiple regions of alpha-actinin interacted with the ICAM-2 peptide.

Conclusions:

  • Alpha-actinin directly binds to the cytoplasmic tail of ICAM-2.
  • The interaction between ICAM-2 and alpha-actinin is mediated by specific amino acid sequences within ICAM-2 and involves multiple domains of alpha-actinin.
  • These findings highlight alpha-actinin's significant role as a molecular linker connecting cell surface adhesion molecules, including ICAM-2, to the actin cytoskeleton.

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