Membrane-proximal α/β stalk interactions differentially regulate integrin activation

The affinity of integrin-ligand interaction is regulated extracellularly by divalent cations and intracellularly by inside-out signaling. We report here that the extracellular, membrane-proximal alpha/beta stalk interactions not only regulate cation-induced integrin activation but also play critical roles in propagating inside-out signaling. Two closely related integrins, alpha IIb beta 3 and alpha V beta 3, share high structural homology and bind to similar ligands in an RGD-dependent manner. Despite these structural and functional similarities, they exhibit distinct responses to Mn2+. Although alpha V beta 3 showed robust ligand binding in the presence of Mn2+, alpha IIb beta 3 showed a limited increase but failed to achieve full activation. Swapping alpha stalk regions between alpha IIb and alpha V revealed that the alpha stalk, but not the ligand-binding head region, was responsible for the difference. A series of alpha IIb/alpha V domain-swapping chimeras were constructed to identify the responsible domain. Surprisingly, the minimum component required to render alpha IIb beta 3 susceptible to Mn2+ activation was the alpha V calf-2 domain, which does not contain any divalent cation-binding sites. The calf-2 domain makes interface with beta epidermal growth factor 4 and beta tail domain in three-dimensional structure. The effect of calf-2 domain swapping was partially reproduced by mutating the specific amino acid residues in the calf-2/epidermal growth factor 4-beta tail domain interface. When this interface was constrained by an artificially introduced disulfide bridge, the Mn2+-induced alpha V beta 3-fibrinogen interaction was significantly impaired. Notably, a similar disulfide bridge completely abrogated fibrinogen binding to alpha IIb beta 3 when alpha IIb beta 3 was activated by cytoplasmic tail truncation to mimic inside-out signaling. Thus, disruption/formation of the membrane-proximal alpha/beta stalk interface may act as an on/off switch that triggers integrin-mediated bidirectional signaling.