Integrin cytoplasmic domains mediate inside-out signal transduction

We analyzed the binding of fibronectin to integrin α₅β₁ in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd ~ 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of α(IIb)β₃ joined to the cytoplasmic domains of α₅β₁. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of α₅β₁ conferred an energy-dependent high affinity state on α(IIb)β₃ in CHO but not K562 cells. Three additional α cytoplasmic domains (α₂, α₆A, α₆B) conferred PAC1 binding in CHO cells, while three others (α(M), α(L), α(v)) did not. In the high affinity α chimeras, cotransfection with a truncated (β₃Δ724) or mutated (β₃(S⁷⁵²→P)) β₃ subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved α subunit GFFKR motif, resulted in high affinity binding of ligands to α(IIb)β₃. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the β subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the α subunit cytoplasmic domain maintains the default low affinity state.