Post-receptor occupancy events in leukocytes during β1 integrin-ligand interactions

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Abstract
Leukocyte adhesion to and subsequent spreading on the endothelium are the initial steps in the migration of these cells to the surrounding tissues. We have investigated the participation of different VLA heterodimers in cell spreading by using the anti‐β1 TS2/16 monoclonal antibody (mAb) which induces a conformational change of different VLA integrin receptors, enabling a high‐affinity interaction with their ligands. Both VLA‐4 and VLA‐5 fibronectin (FN), as well as VLA‐2 collagen (COL) receptors mediated cell spreading and morphological changes. The spreading of U‐937 and α4‐transfected K‐562 cells was induced in both FN and VCAM‐1, suggesting that the morphological changes may be induced by cell‐cell as well as cell‐extracellular matrix (ECM) interactions. Furthermore, the β‐regulated cell spreading on VCAM‐1 and COL took place independently of the VLA‐5 FN receptor function. The enhancing effect on cell attachment induced by anti‐β1 TS2/16 mAb was observed in the presence of different doses of cytochalasin D, whereas cell spreading was abolished. Signal transduction during β1‐stimulated integrin‐ligand interaction was also investigated. We have found the co‐localization of β1 integrins and tyrosine‐phosphorylated proteins during the spreading of U‐937 and α2‐ and α4‐transfected K‐562 cells on both ECM (FN and COL) and cellular (VCAM‐1) ligands. Kinetic studies showed that tyrosine phosphorylation was almost coincident with cellular spreading. The tyrosine phosphorylation of polypeptides of 130 kDa and 77 kDa was triggered in U‐937 cells by the interaction of FN with the VLA‐5 receptor in a high‐affinity conformation. However, no signaling was observed by inducing the high‐affinity state of receptor in the absence of appropriate ligand. These data suggest that tyrosine kinase activation is a post‐receptor occupancy event that might be critical in regulating the adhesive properties of integrins.