Studies on alpha v beta 3/ligand interactions using a [3H]SK&F-107260 binding assay.

The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily that mediates cell attachment on arginine-glycine-aspartic acid (RGD)-containing adhesive proteins. A solid-phase microtiter assay was developed to investigate the binding properties of purified alpha v beta 3, using tritiated [3H]SK&F-107260 as the radiolabeled ligand. alpha v beta 3, purified from human platelets, human placenta, and chicken osteoclasts, bound [3H]SK&F-107260 saturably and specifically. Saturation binding studies using platelet alpha v beta 3 revealed a single class of high affinity binding sites, exhibiting a Kd of 1.44 nM and Bmax of 0.20 mol of [3H]SK&F-107260/mol of alpha v beta 3. [3H]SK&F-107260 binding was inhibited by a variety of RGD-containing peptides and by the snake venom protein echistatin, whereas an RGE-containing peptide and four nonpeptide fibrinogen receptor (alpha IIb beta 3) antagonists failed to do so. This study shows that alpha v beta 3 exhibits distinct ligand specificity from the structurally homologous fibrinogen receptor, alpha IIb beta 3. The relative potencies of the RGD-containing peptides in inhibiting [3H]SK&F-107260 binding to alpha v beta 3 were the same as their relative potencies in inhibiting biotinylated-fibrinogen binding to the receptor. alpha v beta 3 purified from chicken osteoclasts and human placenta bound [3H]SK&F-107260 with similar affinities and displayed the same pharmacological profile as the platelet vitronectin receptor. The alpha v beta 3 antagonists inhibited the attachment of MG63 human osteosarcoma cells or rat osteoclasts to recombinant rat osteopontin. The rank order of potency of the antagonists in the cell adhesion assays was similar to that of the receptor binding assay, suggesting that the purified alpha v beta 3-[3H]SK&F-107260 binding assay is a valid reflection of the ligand binding to alpha v beta 3 on cell systems.