Regulation of integrin affinity on cell surfaces

Abstract

Lymphocyte activation triggers adhesiveness of lymphocyte function-associated antigen-1 (LFA-1; integrin α(L)β(2)) for intercellular adhesion molecules (ICAMs) on endothelia or antigen-presenting cells. Whether the activation signal, after transmission through multiple domains to the ligand-binding αI domain, results in affinity changes for ligand has been hotly debated. Here, we present the first comprehensive measurements of LFA-1 affinities on T lymphocytes for ICAM-1 under a broad array of activating conditions. Only a modest increase in affinity for soluble ligand was detected after activation by chemokine or T-cell receptor ligation, conditions that primed LFA-1 and robustly induced lymphocyte adhesion to ICAM-1 substrates. By stabilizing well-defined LFA-1 conformations by Fab, we demonstrate the absolute requirement of the open LFA-1 headpiece for adhesiveness and high affinity. Interaction of primed LFA-1 with immobilized but not soluble ICAM-1 triggers energy-dependent affinity maturation of LFA-1 to an adhesive, high affinity state. Our results lend support to the traction or translational motion dependence of integrin activation.

Figure 1

Integrin activation states and competition binding assay schematic. (A–D) Integrin activation states. Fab and small molecule inhibitors used in this study are shown. Fabs are shown bound to all integrin conformations they bind, and small molecules are shown with conformations that they stabilize. (A) An inactive integrin in a bent conformation. (B) Disruption of TM domain association induces an extended conformation with a closed headpiece. (C) After extension, the hybrid domain can swing out in the open integrin headpiece with an active βI domain, which is suggested to pull down the αI domain α7 helix by binding of α_L-E310 to the β₂ MIDAS. (D) The α/β I allosteric class of integrin inhibitors are believed to ligate the βI domain and induce integrin extension and the open headpiece, but to block activation of the αI domain. (E) Basis for competition binding assay, based on the crystal structure of the α_L I domain bound to ICAM-1 domains 1 and 2 (Shimaoka et al, 2003b). The LFA-1 I domain is depicted as a molecular surface in grey. Species-specific residues in the TS1/22 epitope are coloured in orange. The ligand-binding MIDAS Mg²⁺ is in red and ICAM-1-binding surface is in green. Binding of ICAM-1 (grey Cα trace) is sterically hindered by TS1/22 binding. Taken from Lu et al (2004). (F) Schematic of the competition binding assay. Radiolabelled TS1/22 Fab at a fixed concentration and varying concentrations of Hi3-ICAM-1 are mixed with LFA-1-expressing cells. After equilibrium binding is reached, cells are pelleted through an oil cushion and bound radioactivity is measured.

Figure 2

Soluble ligand-binding assays of LFA-1 affinity. (A) Saturation ¹²⁵I-TS1/22 Fab binding to T lymphocytes. (B, C) Competitive binding of Hi3-ICAM-1 and ¹²⁵I-TS1/22 Fab to cultured T lymphocytes (B) or K562 transfectants (C). (D, E) ¹²⁵I-Hi3-ICAM-1 saturation binding (D) and competitive binding of Hi3-ICAM-1 and ¹²⁵I-TS1/22 Fab (E) to high affinity (K287C, K294C) LFA-mutant K562 transfectants. (F) ¹²⁵I-Hi3-ICAM-1 saturation binding to high affinity (F265S, F292A) LFA-1-mutant K562 transfectants. (G) Competitive binding of Hi3-ICAM-1 and ¹²⁵I-TS1/22 Fab to GFFKR/GAAKR LFA-1-mutant K562 transfectants. Data are averages±s.e. from two independent triplicate experiments. Curves are best fits to specific or total and nonspecific binding models. Specific binding (S) in (A, D, F) was calculated by subtracting nonspecific (NS) from total binding (T). K_D values were calculated from best-fit curves and are indicated±s.e. (A, D, F) or with ±1 s.e. intervals (B, C, E, G).

Figure 3

Inside–out activation of LFA-1 on T lymphocytes induces integrin priming. Lymphocyte adhesion to ICAM-1 substrates (A–D) and Hi3-ICAM-1 competition binding (E–H) after activation by PMA (A, E), SDF-1α (B, F), crosslinking TCR with OKT3 plus anti-IgG_2a (C, G), or Mn²⁺ activation (D, H). Control was medium (A, B, D–F, H) or mouse IgG_2a plus anti-IgG_2a antibody (C, G). Adhesion results are averages±s.e. (n=3–9) and P values are from two-tailed unpaired t-tests. Activation was for 30 min (A, D), 2 min (B), or 10 min (C). Hi3-ICAM-1 binding data are averages±s.e. (n=2, in triplicates). Control data for (E, F, H) are from Figure 2B. Affinity values were calculated from best-fit curves and indicated with ±s.e. intervals in parentheses. Affinities were compared with control by F-tests. (I) KIM127 and m24 activation epitope exposure after cell activation. Numbers indicated are mean fluorescence intensities and are representative for two independent experiments.

Figure 4

The open LFA-1 headpiece is required for adhesion and high affinity for soluble ligand. (A) Lymphocyte adhesion to ICAM-1 substrate after activation by Fab. Averages±s.e. (n=3–13). (B) Activation epitope exposure after CBR LFA-1/2 Fab activation of LFA-1. Mean fluorescence intensities are indicated and representative of two independent experiments. (C–K) Competition binding of Hi3-ICAM-1 after LFA-1 activation by Fab. Results are averages±s.e. from two independent triplicate experiments. Curves are best fits. Calculated K_D values with s.e. interval are in parentheses. Affinities in one-site binding models were compared with control (Figure 2B) by F-test and P values are shown. If data fit significantly better to a two-site binding model (F-test), P values and results for both receptor populations are shown.

Figure 5

Communication between the open headpiece and the α_L I domain is required for adhesion and high affinity for soluble ligand. (A) m24 and KIM127 activation epitope exposure. Indicated mean fluorescence intensities are representative of two independent experiments. (B, D, F) Competition binding of Hi3-ICAM-1 to T lymphocytes (B, D) and K562 transfectants (F). Data are average±s.e. from two independent triplicate experiments. Curves are best fits and affinities are given with s.e. intervals in parentheses. Affinities were compared to control (Figure 2B) (B, D) or nonactivated K562 transfectants (F) by F-tests and P values are shown. (C, E) T lymphocyte (C, E) and SKW3 cell (C) adhesion to ICAM-1 substrate. n.d., not determined. Results are averages±s.e. of triplicates.

Figure 6

Conversion from primed or intermediate affinity to adhesive high affinity LFA-1 requires cellular energy. (A) T lymphocyte adhesion to ICAM-1 after pretreatment of cells with 2-deoxy-glucose (2-DG) and sodium azide. Shown are averages±s.e. (n=8–9). Mean values were compared by unpaired two-tailed t-tests with Welch's correction (^***P<0.001; NS, not significant). (B–F) Competition binding of Hi3-ICAM-1 to T lymphocytes pretreated with 2-DG and azide and stimulated with various activators. Data are shown as average±s.e. from two independent triplicate experiments. Solid lines are best fits to data and affinities are given with s.e. intervals in parentheses. If data fit significantly better to a two-site binding model (F-test), P values and results for both receptor populations are shown. For comparison, data from Figures 2, 3 and 4 on cells without pretreatment are shown as dashed lines, with affinities and receptor populations in those samples shown after ‘versus’.

Figure 7

Models of integrin activation by inside–out signalling. (A) An inactive bent integrin. (B) The integrin is activated by protein binding to the β cytoplasmic tail, disrupting TM domain association and inducing an extended conformation with a closed or open headpiece. This model proposes equal binding affinity (‘=’) of soluble or immobilized ligand. (C) The translational motion or traction force model suggests that disruption of the TM domain association by cytoskeletal proteins can induce integrin extension, but fails to induce the open headpiece conformation due to considerable flexibility between integrin leg domains. This model proposes that the actin cytoskeleton induces translational motion. When immobilized ligand binds integrin, it resists translation, and force increases (arrows). Force is on pathway with hybrid domain swing out. Force thus acts as an allosteric effector by straightening the β leg, and pulling on the hybrid domain to swing it out. This model predicts substantially higher integrin affinity (‘>’) for ligand on substrates than in solution phase.