Mutation of a Conserved Asparagine in the I-like Domain Promotes Constitutively Active Integrins αLβ2 and αIIbβ3

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The integrin ␣ L ␤ 2 mediates leukocyte adhesion and migration that are required for a functional immune system. It is known that inside-out signaling triggers ␣ L ␤ 2 conformational changes, which affect its ligand-binding affinity. At least three ␣ L ␤ 2 affinity states (low, intermediate, and high) were described. The cytosolic protein talin connects ␣ L ␤ 2 to the actin filament. The talin head domain is also known to activate ␣ L ␤ 2 ligand binding. However, it remains to be determined whether talin promotes an intermediate or high affinity ␣ L ␤ 2 . In this study using transfectants and T cells, we showed that talin induced an intermediate affinity ␣ L ␤ 2 that adhered constitutively to its ligand intercellular adhesion molecule (ICAM)-1 but not ICAM-3. Adhesion to ICAM-3 was induced when an additional exogenous activating agent was included. Similar profiles were observed with soluble ICAMs. In addition, the intermediate affinity ␣ L ␤ 2 induced by talin allowed adhesion and migration of T cells on immobilized ICAMs.Integrins are a large family of heterodimeric type I transmembrane adhesion molecules that serve important roles in many biological processes, including cell growth, migration, differentiation, and apoptosis (1). In humans, 18 integrin ␣ subunits and 8 integrin ␤ subunits form 24 non-covalently associated heterodimers. Integrins have large ectodomains and relatively short cytoplasmic tails with the exception of the ␤ 4 subunit. Integrins are not known to have intrinsic enzymatic activities and signaling is achieved via the recruitment of cytosolic signaling proteins to their cytoplasmic tails.The integrin ectodomain can be segregated into two collective regions referred to as the headpiece and tailpiece ( Fig. 1) (2). The headpiece participates in ligand-binding. Structural studies of ␣ IIb ␤ 3 , ␣ V ␤ 3 , and ␣ 5 ␤ 1 suggest at least three integrin ligand-binding affinity states having different conformations (2-6). The low affinity integrin is represented by an acutely bent structure with a closed headpiece. By contrast, a high affinity integrin adopts an extended conformation with open headpiece derived from hybrid domain displacement. The intermediate affinity integrin conformation is proposed to be a collection of bent structures with open headpieces or extended structures with closed headpieces (3). A recent electron microscopy study of soluble recombinant ␤ 2 integrins reveals three major conformers (7), the bent conformer and two extended conformers with open and closed headpieces.Previously, we reported the requirement of integrin ␣ L ␤ 2 (leukocyte function-associated antigen-1 and CD11aCD18) having different affinity states for the adhesion to its ligands intercellular adhesion molecule-1 (ICAM-1) 3 (CD54) and -3 (CD50) (8). Whereas an intermediate affinity ␣ L ␤ 2 bound ICAM-1, a high affinity ␣ L ␤ 2 was required for ICAM-3 binding. The Inserted (I)-domain (also known as the ␣A domain (9)) is the primary ligand-binding domain of ␣ L ␤ 2 . The order of binding affinities to differe...

Section: Discussionsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 97%

The Cytosolic Protein Talin Induces an Intermediate Affinity Integrin αLβ2

Tang

Puan

et al. 2007

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“…Further, we showed that the conformational changes in ␣L␤2 that are required to bind ICAM-1 and ICAM-3 are different (43). In our previous studies, the adhesion of ␣L␤2 expressing cells to ICAM-3 can be induced by a combination of KIM185 and Mg/EGTA (25,41,44,45). In line with these observations, transfectant expressing wild-type ␣L␤2 only adhered significantly to ICAM-3 when both activating KIM185 and Mg/EGTA were included (Fig.…”

Section: Resultssupporting

confidence: 72%

Disruption of the Integrin αLβ2 Transmembrane Domain Interface by β2 Thr-686 Mutation Activates αLβ2 and Promotes Micro-clustering of the αL Subunits

Vararattanavech¹,

Lin

Torres

et al. 2009

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Integrins are type I heterodimeric cell adhesion molecules that mediate a wide array of biological processes. Integrin bidirectional signaling allows communication between the cell interior with its microenvironment. The integrin transmembrane domains (TMs) are the transducers of activation signal that is relayed from the cytoplasmic domains to the distal ligand binding site located in the ectodomain of the integrin and vice versa. In this study, we showed that the disruption of the ␣L␤2 TMs by mutation of a key interface residue Thr-686 in the ␤2 TM promoted ␣L␤2 activation with ICAMs binding properties that are reminiscent of an intermediate affinity receptor. The activated ␣L␤2 TM mutants, however, showed minimal reactivity with the reporter mAb KIM127 that recognizes a highly extended ␣L␤2. Two models of ␣L␤2 TM interaction were proposed previously. One with GXXXG-type interaction, and another that is based on TM cysteine-scanning analyses. Our data are consistent with a GXXXG-type interaction of the ␣L␤2 TMs. Finally, we observed by FRET analyses that perturbation of the ␣L␤2 TMs by ␤2 Thr-686 mutation facilitated ␣L microcluster formation. This was diminished by linking the ␣L␤2 TMs with a disulfide bond, which served to clasp the TMs. These data suggest that disruption of the TM interface changes ␣L␤2 ligand binding affinity, and it may contribute to ␣L micro-cluster formation.

“…We directly confirmed the formation of the disulfide bond by mass spectrometry and tested the interaction of both large and small ligands to clamped ␣IIb␤3, as well as both soluble and immobilized ligands. We also tested the effects of multiple ␣IIb␤3 activation mechanisms, including Mn 2ϩ and coexpression of normal and mutant ␣IIb with a mutant ␤3 subunit that when coexpressed with normal ␣IIb results in spontaneous fibrinogen binding (N339S) (23). Our data support an important role for receptor extension around the genu for the binding of large soluble ligands induced by some, but not all activation mechanisms.…”

mentioning

confidence: 69%

“…1). We separately generated the ␤3 mutation N339S, which has been shown to constitutively activate ␣IIb␤3 and promote fibrinogen binding (23).…”

mentioning

confidence: 99%

Effects of Limiting Extension at the αIIb Genu on Ligand Binding to Integrin αIIbβ3

Blue

Steinberger

et al. 2010

Structural data of integrin ␣IIb␤3 have been interpreted as supporting a model in which: 1) the receptor exists primarily in a "bent," low affinity conformation on unactivated platelets and 2) activation induces an extended, high affinity conformation prior to, or following, ligand binding. Previous studies found that "clasping" the ␣IIb head domain to the ␤3 tail decreased fibrinogen binding. To study the role of ␣IIb extension about the genu, we introduced a disulfide "clamp" between the ␣IIb thigh and calf-1 domains. Clamped ␣IIb␤3 had markedly reduced ability to bind the large soluble ligands fibrinogen and PAC-1 when activated with monoclonal antibody (mAb) PT25-2 but not when activated by Mn 2؉ or by coexpressing the clamped ␣IIb with a ␤3 subunit containing the activating mutation N339S. The clamp had little effect on the binding of the snake venom kistrin (M r 7,500) or ␣IIb␤3-mediated adhesion to immobilized fibrinogen, but it did diminish the enhanced binding of mAb AP5 in the presence of kistrin. Collectively, our studies support a role for ␣IIb extension about the genu in the binding of ligands of 340,000 and 900,000 M r with mAb-induced activation but indicate that it is not an absolute requirement. Our data are consistent with ␣IIb extension resulting in increased access to the ligand-binding site and/or facilitating the conformational change(s) in ␤3 that affect the intrinsic affinity of the binding pocket for ligand.The platelet ␣IIb␤3 receptor plays an important role in both hemostasis and thrombosis (1). Ligand binding to ␣IIb␤3 is controlled by an activation process that affects the conformation of the receptor and ligand binding, in turn, can also affect the conformation of the receptor (2). Several different conformations of ␣IIb␤3 have been identified based on inferences from biochemical analyses (3), studies employing monoclonal antibodies (4 -7) and electron microscopy (8 -10), comparison of the crystal structures of the liganded ␣IIb␤3 headpiece (11) and the unliganded complete ectodomain (12), and analysis of the unliganded and liganded ectodomain of the related ␣V␤3 receptor (13,14). Receptor extension about the regions encompassing the thigh, genu, and calf-1 domains of ␣IIb and the plexin-semaphorin-integrin (PSI), 2 integrin epidermal growth factor-1 (IEGF-1), and IEGF-2 domains of ␤3 or comparable regions of other integrin receptors has been proposed to play an important role in receptor activation (12,(15)(16)(17)(18), but there is uncertainty about whether this conformational change occurs prior to or after ligand binding (19 -21). Thus, "cross-clasping" the ␣IIb headpiece ␤-propeller domain to the ␤3 IEGF-4 domain in the tail region via a newly engineered disulfide bond prevented the binding of fibrinogen induced by activating mAb in concert with the activating divalent cation Mn 2ϩ , and a similar effect was observed with cross-clasped ␣V␤3 (16). In both cases the loss of ligand binding could be rescued by reducing the cross-clasped receptors with dithiothreitol (DTT). In contr...