Association of the Cytoplasmic Domain of Intercellular‐Adhesion Molecule‐1 with Glyceraldehyde‐3‐Phosphate Dehydrogenase and β‐Tubulin

To elucidate the molecular mechanisms of the transendothelial migration of leukocytes, we attempted to identify the cellular proteins capable of interaction with the cytoplasmic domain of the intercellular adhesion molecule‐1 (ICAM‐1) in a rat brain microvessel endothelial cell line (RBE4 cells). A 27‐amino‐acid synthetic peptide, corresponding to the cytoplasmic domain of rat ICAM‐1, was covalently linked to a Sepharose matrix. Upon affinity chromatography of RBE4 cell cytosol, several ICAM‐1‐interacting proteins were specifically eluted by the soluble peptide. Two of these proteins have been identified by microsequencing as the cytoskeletal protein β‐tubulin and the glycolytic enzyme glyceraldehyde‐3‐phos‐phate dehydrogenase (GraP‐DH). Experiments carried out with purified GraP‐DH or CNBr fragments of GraP‐DH indicated that binding to the ICAM‐1 matrix was mediated by the C‐terminal domain of GraP‐DH, containing the binding site of the cofactor NATL+, and that NATL + could compete with this binding. Using a series of ICAM‐1 C‐terminal truncated peptides, we could demonstrate that (a) the nitric‐oxide‐induced covalent linkage of NATL + to GraP‐DH was impaired by these peptides, (b) the glycolytic activity of GraP‐DH was drastically inhibited by a truncated peptide containing the 15 C‐terminal residues, (c) nitric oxide appeared to prevent this inhibition. Together, our results demonstrate that Gra P ‐DH specifically associates with the isolated ICAM‐1 cytoplasmic domain. Since Gra P ‐DH is known as a microtubule bundling protein, these findings suggest that, in a cellular environment, GraP‐DH may behave as an Actaptor molecule by linking ICAM‐1 to the microtubule network. The role of nitric oxide in the modulation of this interaction deserves further investigation.