Evidence from site-directed mutagenesis that the cytoplasmic domain of thebeta 3 subunit influences the conformational state of the alpha v beta 3 integrin ectodomain

In order to explore the mechanisms leading to conformational changes of the vitronectin receptor alphav beta3 following ligand or divalent cation bind ing, we have investigated the expression of epitopes known as ligand-induce d binding sites (LIBS) on beta3 cytoplasmic tail mutants expressed in CHO c ells. Truncation of the entire beta3 cytoplasmic domain induced constitutiv e LIES exposure on (alphav beta3 and alpha IIb beta3. Deletion of the C-ter minal NITY759 sequence or disruption of the NPLY747 motif by a Y747A' subst itution impaired extracellular conformational changes on alphav beta3 follo wing RGDS, echistatin or Mn2+ binding, whereas the substitutions Y747F, Y75 9A or Y759F allowed normal LIES exposure. Furthermore, metabolic energy dep letion totally prevented Mn2+-dependent LIES exposure, but had only a minor effect on RODS-induced conformational changes. Our results demonstrate tha t the structural integrity of the NPLY747 motif in the beta3 cytoplasmic do main, rather than potential phosphorylation of Tyr(747) Or Tyr(759) is a pr erequisite for conformational changes within the alphav beta3 ectodomain, a nd suggest that two different mechanisms are responsible for RGDS- and Mn2-dependent conformational changes.