Association of the Membrane Proximal Regions of the α and β Subunit Cytoplasmic Domains Constrains an Integrin in the Inactive State

Abstract

The adhesiveness of integrins is regulated through a process termed "inside-out" signaling. To understand the molecular mechanism of integrin inside-out signaling, we generated K562 stable cell lines that expressed LFA-1 (αLβ2) or Mac-1 (α Mβ2) with mutations in the cytoplasmic domain. Complete truncation of the β2 cytoplasmic domain, but not a truncation that retained the membrane proximal eight residues, resulted in constitutive activation of αLβ2 and αMβ2, demonstrating the importance of this membrane proximal region in the regulation of integrin adhesive function. Furthermore, replacement of the αL and β2 cytoplasmic domains with acidic and basic peptides that form an α-helical coiled coil caused inactivation of αLβ2. Association of these artificial cytoplasmic domains was directly demonstrated. By contrast, replacement of the αL and β2 cytoplasmic domains with two basic peptides that do not form an α-helical coiled coil activated αLβ2. Induction of ligand binding by the activating cytoplasmic domain mutations correlated with the induction of activation epitopes in the extracellular domain. Our data demonstrate that cytoplasmic, membrane proximal association between integrin α and β subunits, constrains an integrin in the inactive conformation.