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 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.
Federici
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C
Laboratoire d'Immuno-Pharmacologie Moléculaire, ICGM, CNRS UPR 0415, Paris, France.
Camoin
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L
Hattab
M
M
Strosberg
A D
AD
Couraud
P O
PO
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000187:drug effects / Q000378:metabolism*; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D015398:Signal Transduction; D013379:Substrate Specificity; D014404:Tubulin / Q000378:metabolism*
D010446:Peptide Fragments; D014404:Tubulin; D009243:NAD; D018799:Intercellular Adhesion Molecule-1; D009569:Nitric Oxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases