αvβ3 integrin‐ligand binding is regulated by protein kinase A

Abstract

We have demonstrated that in endothelial cells β1 integrin antagonists inhibited αvβ3 integrin mediated adhesion. Here we have performed vitronectin binding assays on endothelial cells in suspension. Vitronectin binding was inhibited upon β1 integrin clustering, indicating that β1 integrin modulated αvβ3 integrin affinity. Over-expression of talin, a known modulator of integrin affinity, did not abrogate the impact of β1 integrin clustering on αvβ3 integrin-ligand binding. Rather, β1 integrin clustering resulted in a two-fold increase in PKA activity and β3 integrin Ser phosphorylation. PKA inhibition rescued cell adhesion when β1 integrin-ligand binding was blocked and inhibited β3 integrin Ser phosphorylation. β3 integrin contains a Ser residue at position 752. We mutated it to Ala (β3S752A) or Asp (β3S752D). Cells expressing wild-type or β3S752A integrin attached robustly to ligand. Cells expressing β3S752D integrin did not. Since the β3 cytoplasmic tail lacks a PKA consensus site, we hypothesized that PKA regulates β3 integrin Ser phosphorylation indirectly through protein phosphatase 1 (PP1). Indeed, blocking PP1 activity inhibited cell adhesion and increased β3 integrin Ser phosphorylation. These results indicate a novel mechanism by which clustered β1 integrin negatively modulates αvβ3 integrin-ligand binding via activation of PKA and inhibition of PP1 activity. Supported by NCI.