A HIGH-AFFINITY CONFORMATIONAL STATE ON VLA INTEGRIN HETERODIMERS INDUCED BY AN ANTI-BETA-1 CHAIN MONOCLONAL-ANTIBODY

The VLA integrin subfamily includes receptors for extracellular matrix proteins as well as receptors involved in cell-cell adhesive interact ions. We have previously described the up-regulation of VLA integrin-m ediated cell attachment to different ligands by the anti-beta1 TS2/16 monoclonal antibody (mAb) (Arroyo, A. G., Sanchez-Mateos, P., Campaner o, M. R., Martin-Padura, I., Dejana, E., and Sanchez-Madrid, F. (1992) J. Cell Biol. 117, 659-670). In this report, we have investigated the mechanism involved in this regulatory effect. The anti-beta1-mediated regulatory effect on cell adhesion did not require ''de novo'' protei n synthesis, since it was not affected by pretreatment with either cyc loheximide or actinomycin D. To quantitate the effect of the regulator y anti-beta1 TS2/16 mAb on the affinity of VLA-5 for its ligand, an RG D-containing fragment of fibronectin (FN80), we performed binding stud ies of radiolabeled soluble FN80 to U-937 cells. The affinity of VLA-5 for FN80 was enhanced about 4-fold in the presence of TS2/16 mAb (K(d ) = 0.98 +/-0.07 muM) compared to the functionally irrelevant anti-bet a1 Alex 1/4 mAb (K(d) = 4.23 +/- 0.92 muM), whereas no alteration in t he number of binding sites was observed. Indeed, the anti-beta1 TS2/16 mAb is inducing this high affinity state on VLA heterodimers by a dir ect change on the conformation of these receptors as demonstrated by a ffinity chromatography analysis using extracellular matrix proteins co valently bound to Sepharose. The yield of VLA-5 fibronectin receptor b ound to FN80-Sepharose columns was strongly increased upon treatment o f U-937 cell lysates with mAb TS2/16. Moreover, higher concentrations of EDTA were required for eluting the VLA-5 integrin from this matrix. This up-regulatory effect was also observed with F(ab')2 and Fab frag ments of the anti-beta1 TS2/16 mAb, and was also exerted on the purifi ed VLA-5 receptor. Similarly, the yield of VLA-2 retained on a collage n I-Sepharose column was dramatically increased by pretreatment of A37 5 melanoma cell lysates with the mAb TS2/16. Altogether, these results indicate that the interaction of VLA beta1 heterodimers with their li gands can be regulated by switching between differently active conform ations inherent to the alphabeta1 receptors.