Regulation of integrin activation by intracellular and extracellular signaling. (A and B) ΔG of each integrin conformational state for integrin headpiece or ectodomain preparations or intact cell surface integrins measured here for α₄β₁ (A) and here and previously for α₅β₁ (B; Li et al., 2017). (C) Energy landscape comparisons for intact α₅β₁ and α₄β₁ on Jurkat, Thp1, and KA4 cells. (D) Schematic illustration of integrin activation regulated by intracellular protein binding to integrin cytoplasmic tails and application of cytoskeletal force. Ovals represent inhibitors and stars represent adaptors. F represents tensile force exerted across ligand–integrin–adaptor complexes by the cytoskeleton and resisted by immobilized ligand. Distances in the force-bearing pathway between the ligand-binding site and the C terminus of the β-tail domain known from structures and molecular dynamics are shown with arrows (x₁, x₂, and x₃). (E–G) Population of integrin states that mediate cell adhesion in absence (E) or presence of force (F and G). Colors in the key encode the population of integrin states that can mediate cell adhesion (i.e., the sum of adaptor and ligand bound states over all three integrin conformational states). >99% of such adaptor- and ligand-bound integrins are in the EO conformation (Li and Springer, 2017). Ligands are used at concentrations equal to $KdEO$_. Adaptors are assumed to bind to EC and EO and not BC states and are used at a concentration equal to the adaptor K_d in G. Rectangles with white dashed lines show the range of ΔG values found for integrins α₄β₁ and α₅β₁ on different cell types. (E) Induction of cell adhesion by an increase in adaptor concentration is insensitive to adaptor concentration. (F) Presence of a moderate 1.5 pN cytoskeletal force gives sensitive integrin activation by variation in adaptor concentration. (G) Variation in force with a fixed adaptor concentration gives ultrasensitive regulation of integrin adhesiveness. Population of adaptor and ligand bound states was calculated according to Eq. S20 as previously described (Li and Springer, 2017).