Contrasting roles for domain 4 of VCAM-1 in the regulation of cell adhesion and soluble VCAM-1 binding to integrin α₄β₁

Cell adhesion mediated by the interaction between integrin α₄β₁ and VCAM-1 is important in normal physiologic processes and in inflammatory and autoimmune disease. Numerous studies have mapped the α₄β₁ binding sites in VCAM-1 that mediate cell adhesion; however, little is known about the regions in VCAM-1 important for regulating soluble binding. In the present study, we demonstrate that 6D VCAM-1 (an alternatively spiked isoform of VCAM-1 lacking Ig-like domain 4) binds α₄β₁ with a higher relative affinity than does the full-length form of VCAM-1 containing 7 Ig-like extracellular domains (7D VCAM-1). In indirect binding assays, the EC ₅₀ of soluble 6D VCAM-1 binding to α₄β ₁ on Jurkat cells (in 1 mM MnCl₂) was 2 × 10 ^-9 M, compared with 7D VCAM-1 at 11 × 10^-9 M. When used in solution to inhibit α₄β₁ mediated cell adhesion, the IC₅₀ of 6D VCAM-1 was 13 × 10^-9 M, compared with 7D VCAM-1 measured at 150 × 10^-9 M. Removal of Ig-like domains 4,5, or 6, or simply substituting Asp³²⁸ in domain 4 of 7D VCAM-1 with alanine, caused increased binding of soluble 7D VCAM-1 to α₄β₁. In contrast, cells adhered more avidly to 7D VCAM-1 under shear force, as it induced cell spreading at lower concentrations than did 6D VCAM-1. Finally, soluble 6D VCAM-1 acts as an agonist through α₄β₁ by augmenting cell migration and inducing cell aggregation. These results indicate that the domain 4 of VCAM-1 plays a contrasting role when VCAM-1 is presented in solution or as a cell surface-expressed adhesive substrate.