Superactivation of integrin alpha v beta 3 by low antagonist concentrations

Integrins are implicated in cell adhesion, migration and homeostasis, An im portant feature is their ability to adopt different affinity states that ca n be regulated by a variety of intra- and extracellular factors. To study a ffinity modulation of the integrin ectodomain by extracellular factors, we produced a soluble recombinant form of mouse integrin alphav beta3 in a mam malian expression system and isolated it to purity. We show that the two tr ansmembrane truncated integrin subunits stably associate to form a function al receptor, soluble recombinant alphav beta3, The affinity of this recepto r for its ligands vitronectin, fibronectin and fibrinogen can be modulated by the divalent cations magnesium, calcium and manganese, Most importantly, me found that a cyclic RGD-peptide has a biphasic effect on rs alphav beta 3 and native purified alphav beta3, with an antagonistic phase at high conc entrations, and an agonistic phase at low concentrations. This integrin sup eractivation by low antagonist concentrations is shown in binding of sr alp hav beta3 to immobilized ligands by ELISA, and in adhesion of cells that ex press the chimaeric integrin ligand KISS31 to immobilized rs alphav beta3 a nd native purified alphav beta3, Our results indicate that low concentratio ns of the ligand mimetic cyclo-RGD can result in superactivation of the ext racellular domain of integrin alphav beta3 to a comparable level as activat ion by manganese.