Real Time Analysis of the Affinity Regulation of α4-Integrin: THE PHYSIOLOGICALLY ACTIVATED RECEPTOR IS INTERMEDIATE IN AFFINITY BETWEEN RESTING AND Mn2+ OR ANTIBODY ACTIVATION

This work examines the affinity of alpha(4)beta(1)-integrin and whether affinity regulation by G protein-coupled receptor (GPCR) and chemokines receptors is compatible with cell adhesion mediated between alpha(4)-integrin and vascular cell adhesion molecule-1. We used flow cytometry to examine the binding of a fluorescent derivative of an LDV peptide (Chen, L. L., Whitty, A., Lobb, R. R., Adams, S. P., and Pepinsky, R. B. (1999) J. Biol. Chem. 274, 13167-13175) to several cell lines and leukocytes with alpha4-integrin ranging from about 2,000 to 100,000 sites/cell. The results support the idea that alpha-integrins exhibit multiple affinities and that affinity changes are regulated by the dissociation rate and conformation. The affinity varies by 3 orders of magnitude with the affinity induced by binding mAb TS2/16 plus Mn2+ > Mn-2+ ' TS2/16 > activation because of occupancy of GPCR or chemokines receptor > resting receptors. A significant fraction of the receptors respond to the activating process. The change in alpha4-integrin affinity and the corresponding change in off rates mediated by GPCR receptor activation are rapid and transient, and their duration depends on GPCR desensitization. The affinity changes mediated by IgE receptor or interleukin-5 receptor persist longer. It appears that the physiologically active state of the alpha4-integrin, determined by inside-out signaling, has similar affinity in several cell types.