Association of the cytoplasmic domain of intercellular-adhesion molecule-1 with glyceraldehyde-3-phosphate dehydrogenase and beta-tubulin.

Abstract

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 beta-tubulin and the glycolytic enzyme glyceraldehyde-3-phosphate 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 NAD+, and that NAD+ 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 NAD+ 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 GraP-DH specifically associates with the isolated ICAM-1 cytoplasmic domain. Since GraP-DH is known as a microtubule bundling protein, these findings suggest that, in a cellular environment, GraP-DH may behave as an adaptor molecule by linking ICAM-1 to the microtubule network. The role of nitric oxide in the modulation of this interaction deserves further investigation.