Contrasting roles for domain 4 of VCAM-1 in the regulation of cell adhesion and soluble VCAM-1 binding to integrin alpha4beta1.
Cell adhesion mediated by the interaction between integrin alpha4beta1 and VCAM-1 is important in normal physiologic processes and in inflammatory and autoimmune disease. Numerous studies have mapped the alpha4beta1 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 spliced isoform of VCAM-1 lacking Ig-like domain 4) binds alpha4beta1 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 EC50 of soluble 6D VCAM-1 binding to alpha4beta1 on Jurkat cells (in 1 mM MnCl2) was 2 x 10(-9) M, compared with 7D VCAM-1 at 11 x 10(-9) M. When used in solution to inhibit alpha4beta1 mediated cell adhesion, the IC50 of 6D VCAM-1 was 13 x 10(-9) M, compared with 7D VCAM-1 measured at 150 x 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 alpha4beta1. 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 alpha4beta1 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.
Encysive Pharmaceuticals Incorporated, Houston, TX 77030, USA. dwoodside@encysive.com
Darren G
DG
Woodside
Ronda M
RM
Kram
Jason S
JS
Mitchell
Tracie
T
Belsom
Matthew J
MJ
Billard
Bradley W
BW
McIntyre
Peter
P
Vanderslice
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002448:Cell Adhesion / Q000502:physiology*; D002449:Cell Aggregation / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D004247:DNA / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D039041:Integrin alpha4beta1 / Q000378:metabolism*; D018799:Intercellular Adhesion Molecule-1 / Q000502:physiology; D019169:Jurkat Cells; D007700:Kinetics; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D019010:Vascular Cell Adhesion Molecule-1 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*
D039041:Integrin alpha4beta1; D011994:Recombinant Proteins; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D004247:DNA