Structural Requirements for Activation in αIIbβ3 Integrin

Abstract

Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant ␣IIb␤3 integrins that are constrained specifically in either a bent or an extended conformation. These mutant ␣IIb␤3 integrins were expressed in mammalian cells, and fibrinogen binding to these cells was examined. The bent integrins were created through the introduction of artificial disulfide bridges in the ␤-head/␤-tail interface. Cells expressing bent integrins all failed to bind fibrinogen unless pretreated with DTT to disrupt the disulfide bridges. The extended integrins were created by introducing N-glycosylation sites in amino acid residues located close to the ␣-genu, where the integrin legs fold backward. Among these mutants, activation was maximized in one integrin with an N-glycosylation site located behind the ␣-genu. This extension-induced activation was completely blocked when the swing-out of the hybrid domain was prevented. These results suggest that the bent and extended conformers represent low affinity and high affinity conformers, respectively, and that extension-induced activation depends on the swing-out of the hybrid domain. Taken together, these results are consistent with the current hypothesis that integrin affinity is regulated by the switchblade-like movement of the integrin legs.Integrin-mediated bidirectional signaling is closely associated with the structural rearrangement of integrin itself. During inside-out signaling, talin has been shown to bind to the ␤ cytoplasmic tail and to disrupt the endogenous interaction between the ␣ and ␤ cytoplasmic tails (1, 2). The dissociation of the two tails induces a structural rearrangement of the extracellular domains increasing the affinity to the ligand. During outside-in signaling, ligand binding in turn induces the structural rearrangement of the extracellular domains. This structural change propagates through the plasma membrane to separate the cytoplasmic tails, providing binding sites for numerous cytoplasmic proteins (3). Thus, the two structures flanking the plasma membrane affect each other. This structural rearrangement can be detected using a group of monoclonal antibodies (mAbs) that bind preferentially to the ligand-bound form (4). These anti-LIBS 2 (ligand-induced binding site) mAbs have been used not only to investigate the activation status of a specific integrin but also to activate it (5). However, without information on the actual three-dimensional structure, it is impossible to determine the specific conformation recognized by each anti-LIBS mAb.The first observation of the actual three-dimensional structure of integrin was made using conventional electron ...

Modulation of integrin activation and signaling by α1/α1′-helix unbending at the junction

“…Glycan wedge mutations that enforce and disulfide mutations that restrain the hybrid domain swinging out activated and deactivated integrin, respectively (Luo et al, 2003;Kamata et al, 2010). In addition, mutations enforcing integrin extension but restraining the swing-out of the hybrid domain did not activate integrin (Kamata et al, 2010). A recent study showed that talin1 alone only induces integrin extension with intermediate affinity for b2 integrin, whereas both talin1 and kindlin-3 are required to induce the open headpiece, high-affinity conformation (Lefort et al, 2012).…”

“…Mutations that favor the downward movement of the b6-a7 loop and a7-helix constitutively activated integrin for ligand binding (Luo et al, 2004b;Yang et al, 2004;Cheng et al, 2007). Glycan wedge mutations that enforce and disulfide mutations that restrain the hybrid domain swinging out activated and deactivated integrin, respectively (Luo et al, 2003;Kamata et al, 2010). In addition, mutations enforcing integrin extension but restraining the swing-out of the hybrid domain did not activate integrin (Kamata et al, 2010).…”

“…Disulfide bonds introduced into the domain interfaces inactivate integrin by restraining extension, headpiece opening, or leg separation (supplementary material Fig. S5A-D) (Takagi et al, 2002;Blue et al, 2010;Kamata et al, 2010;Wang et al, 2010;Wang et al, 2011;Kamata, 2012). In contrast, mutations that disturb the domain interfaces all constitutively activate integrin by enforcing extension, tail separation or headpiece opening (supplementary material Fig.…”

Intact αIIbβ3 Integrin Is Extended after Activation as Measured by Solution X-ray Scattering and Electron Microscopy

“…However, the postulated "dead bolt" interaction that prevents integrin extension buries an insignificant surface area of only 40 Å 2 in ␣ V ␤ 3 (23) and is not present in ␣ IIb ␤ 3 or ␣ x ␤ 2 , and its mutation in ␣ V ␤ 3 and ␣ IIb ␤ 3 has no effect on their ligand binding capacities (10,24). Disulfide bonds in multiple locations to favor the bent conformation or closed headpiece and glycan wedges to stabilize extension and headpiece opening all support a requirement for integrin extension and headpiece opening for activation and the insufficiency of the bent conformation (11,(25)(26)(27).…”

αIIbβ3 variants defined by next-generation sequencing: Predicting variants likely to cause Glanzmann thrombasthenia

“…Sixth, 3D molecular models have been built based on crystallographic and NMR data to analyze the effects of novel amino acid substitutions on receptor structure and function and the generation of alloantigens (15,(46)(47)(48)(49)(50)(51)(52)(53). The data from these models and assessments of the severity of the amino acid change in the variants have the potential to aid in predicting whether a novel variant is likely to affect receptor function and immunogenicity (54)(55)(56)(57)(58)(59).…”