Association of the Cytoplasmic Domain of Intercellular‐Adhesion Molecule‐1 with Glyceraldehyde‐3‐Phosphate Dehydrogenase and β‐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-I (ICAM-1) in a rat brain microvessel endothelial cell line (RBE4 cells). A 27-aminoacid 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 P-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-oxideinduced 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 associate! 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.

RKIKK Motif in the Intracellular Domain Is Critical for Spatial and Dynamic Organization of ICAM-1: Functional Implication for the Leukocyte Adhesion and Transmigration

“…Leukocyte TEM is inhibited after the inactivation of endothelial cells' Rho protein function (Adamson et al, 1999), thereby suggesting a proactive role for ICAM-1 signaling/cytoskeletal interactions in facilitating TEM. In agreement with these lines, the intracellular domain of ICAM-1 can bind to a variety of molecules, such as ␣-actinin (Carpen et al, 1992), ␤-tubulin, GAPDH (Federici et al, 1996), and ezrin (Heiska et al, 1998). These proteins all have the potential to perform signaling functions.…”

Evidence of a Functional Role for Interaction between ICAM-1 and Nonmuscle α-Actinins in Leukocyte Diapedesis

“…Although ICAM-1 has been reported to bind several intracellular proteins such as ␣-actinin (11), ␤-tubulin, GAPDH (12), and ERM protein (9), this is the first evidence of a functional role for an intracellular ICAM-1 partner. Ezrin has been considered as the leading candidate.…”

Immunolocalization of the exocytosis-sensitive phosphoprotein, PP63/parafusin, in Paramecium cells using antibodies against recombinant protein

“…This also includes structure formation, e.g., formation of lens crystallin (Piatigorsky and Wistow 1989;Wistow 1993) or association with cytoskeletal elements (Frederici et al 1996). In muscle, PGM can associate with desmosomes ), extra-junctional acetylcholine receptors (Belkin and Burridge 1994) or dystrophin (Moiseeva et al 1996).…”