Residues within a conserved amino acid motif of domains 1 and 4 of VCAM-1 are required for binding to VLA-4.

Abstract

Abstract. Vascular cell adhesion molecule 1 (VCAM-1), a member of the Ig superfamily originally identified on activated endothelium, binds to the integrin very late antigen-4 (VLA-4), also known as ot4~l or CD49d/CD29, to support cell-cell adhesion. Studies based on cell adhesion to two alternatively spliced forms of VCAM-1 or to chimeric molecules generated from them and intercellular adhesion molecule-1 (ICAM-1) have demonstrated two VLA-4 binding sites on the predominate form of VCAM-1. Here, we studied VLA-4-dependent adhesion of the lymphoid tumor cell line Ramos to cells expressing wild type and mutant forms of VCAM-1. Results based on domain deletion mutants demonstrated the existence and independence of two VLA-4-binding sites located in the first and fourth domains of VCAM-1. Results based on amino acid substitution mutants demonstrated that residues within a linear sequence of six amino acids found in both domain 1 and 4 were required for VLA-4 binding to either domain. Five of these amino acids represent a conserved motif also found in ICAM domains. We propose that integrin binding to these Ig-like domains depends on residues within this conserved motif. Specificity of integrin binding to Ig-like domains may be regulated by a set of nonconserved residues distinct from the conserved motif.

Contrasting Roles for Domain 4 of VCAM-1 in the Regulation of Cell Adhesion and Soluble VCAM-1 Binding to Integrin α4β1

“…From two experiments, the EC 50 range of cell adhesion for 7D VCAM-1(Ig) was 50 -56 ϫ 10 Ϫ12 M, and the EC 50 of cell adhesion for 6D VCAM-1(Ig) was 50 -62 ϫ 10 Ϫ12 M. Equal capture of VCAM-1 was confirmed by ELISA with biotinylated VCAM-1 mAb 1.4C3 (Fig. 3, dotted line), which recognizes an epitope within domain 2 of VCAM-1 in both 7D and 6D VCAM-1 (27). No significant adhesion was seen to control wells coated with BSA (data not shown).…”

“…Also, removal of Ig-like domains COOH-terminal to domain 3 or simply substituting Asp 328 with Ala increases the relative affinity of 7D VCAM-1 for ␣ 4 ␤ 1 . Previous functional and structural studies of the two NH 2 -terminal domains of VCAM-1 (domains 1 and 2) have demonstrated that the primary ␣ 4 ␤ 1 binding site in domain 1 resides in a solvent-exposed loop (the C-D loop, which contains the essential residue Asp 40 ) (25,27,29,40,41). A secondary "synergy" site, the CЈ-E loop-E strand, is located in the upper face of domain 2 (30), spatially close to the C-D loop of domain 1 (40,41).…”

“…Although integrin ␣ 4 ␤ 1 can bind to domain 1 or domain 4 of VCAM-1 (25)(26)(27) to promote cell adhesion, binding is primarily mediated by residues within the NH 2 -terminal domains in the intact, full-length 7D VCAM-1 (28,29). An ␣ 4 ␤ 1 binding "footprint" has been characterized within VCAM-1 domains 1 and 2.…”

“…However, as a soluble ligand, 2D VCAM-1 binds ␣ 4 ␤ 1 with a lower apparent affinity relative to full-length 7D VCAM-1 (31). This could be due to a second potential ␣ 4 ␤ 1 binding site within domain 4 of 7D VCAM-1 (25)(26)(27). To address this, we generated a number of VCAM-1 constructs, including fulllength 7D VCAM-1 and the alternatively spliced 6D VCAM-1, and tested their binding to integrin ␣ 4 ␤ 1 .…”

“…For FLAG-tagged VCAM-1 constructs, either anti-VCAM-1 mAb 1.4C3 (Chemicon International) or anti-FLAG mAb M2 (Sigma-Aldrich) was incubated with VCAM-1 bound cells for 30 min at 4°C. Monoclonal Ab 1.4C3 recognizes both 6D and 7D VCAM-1 to a similar extent (27). Cells were then washed and incubated with FITCconjugated goat anti-mouse Ab (BioSource International).…”