TRANSMEMBRANE-TRUNCATED ALPHA-V-BETA-3 INTEGRIN RETAINS HIGH-AFFINITYFOR LIGAND-BINDING - EVIDENCE FOR AN INSIDE-OUT SUPPRESSOR

The molecular mechanisms of alpha v beta 3 integrin affinity regulatio n have important biological implications in tumour development, wound repair and angiogenesis. We expressed, purified and characterized reco mbinant forms of human alpha v beta 3 (r-alpha v beta 3) and compared the activation state of these with alpha v beta 3 in its cellular envi ronment. The ligand specificity and selectivity of recombinant full-le ngth and double transmembrane truncations of r-alpha v beta 3 cloned i n BacPAK6 vectors and expressed in Sf9 and High Five insect cells were compared with those of native placental alpha v beta 3 and the recept or in situ on the cell surface. r-alpha v beta 3 integrins were purifi ed by affinity chromatography from detergent extracts of cells (full-l ength), and from the culture medium of cells expressing double-truncat ed r-alpha v beta 3. r-alpha v beta 3 had the same epitopes, ligand-bi nding specificities, bivalent cation requirements and susceptibility t o RGD-containing peptides as native alpha v beta 3. On M21-L4 melanoma cells, alpha v beta 3 mediated binding to vitronectin, but not to vit ronectin unless activated with Mn2+. Non-activated alpha IIb beta 3 in tegrin as control in M21-L-IIb cells had the opposite profile, mediati ng binding to fibrinogen, but not to vitronectin unless activated with Mn2+. Thus these receptors had moderate to low ligand affinity. In ma rked contrast, purified alpha v beta 3 receptors, with or without tran smembrane and cytoplasmic domains, were constitutively of high affinit y and able to bind strongly to vitronectin, fibronectin and fibrinogen under physiological conditions. Our data suggest that, in contrast wi th the positive regulation of alpha IIb beta 3 in situ, intracellular controls lower the affinity of alpha v beta 3, and the cytoplasmic dom ains may act as a target for negative regulators of alpha v beta 3 act ivity.