The final steps of integrin activation: the end game

Abstract

Cell-directed changes in the ligand-binding affinity ('activation') of integrins regulate cell adhesion and migration, extracellular matrix assembly and mechanotransduction, thereby contributing to embryonic development and diseases such as atherothrombosis and cancer. Integrin activation comprises triggering events, intermediate signalling events and, finally, the interaction of integrins with cytoplasmic regulators, which changes an integrin's affinity for its ligands. The first two events involve diverse interacting signalling pathways, whereas the final steps are immediately proximal to integrins, thus enabling integrin-focused therapeutic strategies. Recent progress provides insight into the structure of integrin transmembrane domains, and reveals how the final steps of integrin activation are mediated by integrin-binding proteins such as talins and kindlins.

Figure 1. The structure of the αIIbβ3 integrin transmembrane complex enables inside–out signal transduction

The models depicted are based on the average of an ensemble of 20 calculated simulated annealing NMR structures (Protein data bank identifier 2K9J). a | Sequences of the αIIb and β3 integrin transmembrane domains. The membrane- embedded segments, as assessed by NMR spectroscopy of integrins in phospholipid bicelles, are highlighted in blue. b | Ile966–Arg995 of αIIb integrin and Ile693–Asp723 of β3 integrin adopt well-structured conformations with a predicted crossing angle of 25°. c | Rotating the model in part b by 90° reveals the two discrete elements that mediate the principal interaction of the transmembrane domains. The β3 (left) or αIIb (right) integrin transmembrane domains are depicted as space-filling models, with the ribbon structure in the middle. Basic residues are blue and acidic residues are red. The association of α- and β-integrin transmembrane domains, through packing of Gly residues in the outer membrane leaflet, forms the outer membrane clasp. The novel assembly in the inner membrane leaflet extending into the membrane–cytosol interface forms the inner membrane clasp. d | The outer membrane clasp. Gly972 and Gly976 of αIIb integrin and Gly708 of β3 integrin are shown as atoms that form holes into which side chains from the apposing space filling model of β3 integrin pack (left). Gly708 of β3 integrin forms a hole into which αIIb integrin side chains pack (right). e | The αβ-integrin transmembrane interaction is stabilized by interhelical packing mediated by Phe992–Phe993 of αIIb integrin and then the electrostatic interaction of Arg995 of αIIb integrin with Asp723 of β3 integrin to form the inner membrane clasp. The left panel depicts a space-filling model of the β3 integrin transmembrane domain in which Asp723 of β3 integrin is shown in red and space-filling models of the Phe992, Phe993 and Arg995 side chains of αIIb integrin are shown. The right panel depicts a space-filling model of the αIIb integrin transmembrane domain in which Arg995 of αIIb integrin is shown in blue and space-filling models of the Trp715, Ile719 and Asp723 side chains of β3 integrin are shown.

Figure 2. An affinity-capture method to study transmembrane domain interactions

Use of an affinity-capture method reveals the preferential interaction of αIIb and β3 integrin transmembrane domains (TMDs). a | The transmembrane domain tail bait and prey constructs are depicted. The bait consists of the transmembrane and cytoplasmic domain fused to a FLAG tag (for detection), with a signal peptide at the amino terminus and a tandem affinity purification (TAP) tag at the carboxy terminus. The transmembrane and cytoplasmic domains of the prey are joined at the N terminus to the extracellular domain of an irrelevant type 1 membrane protein, such as the TAC subunit of the interleukin 2 receptor. b | Chinese hamster ovary cells are transiently transfected with baits and preys, cells are lysed and the bait is rapidly and efficiently captured through its TAP tag. Capture of the TAP tag with calmodulin beads is depicted. c | Bound preys are detected by western blotting using an anti-TAC antibody. SP, signal peptide.

Figure 3. Activators, such as talins and kindlins, bind to integrins to cause their activation

a | In a direct model of integrin activation, both activators (A1 and A2) bind simultaneously to the integrin tail and, together, modify or disrupt the inner membrane clasp. Other proteins might be involved. In the other two general models, A1 is the primary activator and A2 is an ‘enabler’. b | In an indirect model, A2 regulates a signalling event (for example, synthesis of co-factors) that enables the activator (A1) to bind β-integrin and induce activation. c | In a displacement of an inhibitor model, A2–β-integrin binding displaces an inhibitor of A1, enabling A1 to bind and activate the integrin.

Figure 4. A road map from thrombin receptors to αIIbβ3 integrin activation

The schematic represents the minimal elements of one pathway of αIIbβ3 integrin activation by thrombin receptors, which were identified through the synthetic reconstruction of pathway components in Chinese hamster ovary cells and studies of gene-targeted platelets. Thrombin cleavage or ligand occupancy of the thrombin receptor proteinase-activated receptor 1 (PAR1; also known as F2R) in human platelets, or PAR4 receptors in mouse platelets, stimulates phospholipid hydrolysis, which results in the generation of inositol trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ stimulates an increase in cytosolic free Ca²⁺, activating Ca²⁺- and DAG-regulated (CALDAG-GEFI; also known as RASGRP1), which in turn converts its encoded protein, RAP1, from a GDP-bound to an active GTP-bound form. Ca²⁺ and DAG also activate certain protein kinase C (PKC) isoforms, including PKCα, which among other actions may facilitate the activation of CALDAG-GEFI. Activation of RAP1 leads to recruitment of its effector, RAP1–GTP-interacting adaptor molecule (RIAM; also known as APBB1IP), and its binding partner, talin 1, to the plasma membrane. This enables talin binding to the β3 integrin tail and talin-induced activation of αIIbβ3 integrin. Kindlin 3 plays a crucial role in this process, but because its mechanistic role is uncertain, it is not depicted here.