ASSOCIATION OF THE CYTOPLASMIC DOMAIN OF INTERCELLULAR-ADHESION MOLECULE-1 WITH GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND BETA-TUBULIN

To elucidate the molecular mechanisms of the transendothelial migratio n of leukocytes, we attempted to identify the cellular proteins capabl e of interaction with the cytoplasmic domain of the intercellular adhe sion molecule-1 (ICAM-1) in a rat brain microvessel endothelial cell l ine (RBE4 cells). A 27-amino-acid synthetic peptide, corresponding to the cytoplasmic domain of rat ICAM-1, was covalently linked to a Sepha rose matrix, Upon affinity chromatography of RBE4 cell cytosol, severa l 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 gl yceraldehyde-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-ter minal truncated peptides, we could demonstrate that (a) the nitric-oxi de-induced covalent linkage of NAD(+) to GraP-DH was impaired by these peptides, (b) the glycolytic activity of GraP-DH was drastically inhi bited by a truncated peptide containing the 15 C-terminal residues, (c ) nitric oxide appeared to prevent this inhibition. Together, our resu lts demonstrate that GraP-DH specifically associates with the isolated ICAM-1 cytoplasmic domain. Since GraP-DH is known as a microtubule bu ndling protein, these findings suggest that, in a cellular environment , GraP-DH may behave as an adaptor molecule by linking ICAM-1 to the m icrotubule network. The role of nitric oxide in the modulation of this interaction deserves further investigation.