Ligand Binding Promotes the Entropy-driven Oligomerization of Integrin αIIbβ3

Hantgan, Roy R.; Lyles, Douglas S.; Mallett, T. Conn; Rocco, Mattia; Nagaswami, Chandrasekaran; Weisel, John W.

doi:10.1074/jbc.m208869200

Cited by 40 publications

(49 citation statements)

References 76 publications

(131 reference statements)

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“…Whereas our studies do not specifically address head separation, other results mitigate against it. The ␣ and ␤ subunits could not be distinguished from one another in EM studies that supported head separation (6), whereas higher resolution EM studies in which the ␣ and ␤ subunits could be distinguished showed no evidence for ligand-stimulated head separation (5), in agreement with many other EM studies (18)(19)(20)(21). Furthermore, binding of cyclic RGD peptides stabilizes rather than destabilizes ␣ and ␤ subunit association (7,22,23), and binding of the Arg moiety of RGD to the ␣ subunit and the Asp moiety to the ␤ subunit across the intersubunit interface would be destabilized by head separation.…”

Section: Discussionsupporting

confidence: 76%

Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand

Luo

Springer

Takagi

2003

Proc. Natl. Acad. Sci. U.S.A.

146

186

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The affinity of the extracellular domain of integrins for ligand is regulated by conformational changes signaled from the cytoplasm. Alternative types of conformational movement in the ligandbinding headpiece have been proposed. In one study, electron micrograph image averages of the headpiece of integrin aV␤3 show two different conformations. The open conformation of the headpiece is present when a ligand mimetic peptide is bound and differs from the closed conformation in the presence of an obtuse angle between the ␤3 subunit hybrid and I-like domains. We tested the hypothesis that opening of the hybrid-I-like domain interface increases ligand-binding affinity by mutationally introducing an N-glycosylation site into it. Both ␤3 and ␤1 integrin glycan wedge mutants exhibit constitutively high affinity for physiological ligands. The data uniquely support one model of integrin activation and suggest that movement at the interface with the hybrid domain pulls down the C-terminal helix of the I-like domain and activates its metal ion-dependent adhesion site, analogously to activation of the integrin I domain.I ntegrins are a family of Ϸ25 cell adhesion molecules that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions and govern migration and anchorage of almost all kinds of cells. Integrins are noncovalently associated ␣␤ subunit heterodimers. Each subunit contains a large N-terminal extracellular domain, a transmembrane segment, and a cytoplasmic C-terminal tail. One unique aspect of integrin function is that the affinity for biological ligand can be up-regulated by a process termed inside-out signaling (1). This is particularly important during activation of leukocytes and platelets, where integrins are converted to high-affinity receptors in Ͻ1 s. It has been suggested that this rapid affinity up-regulation is accomplished by a conformational change in integrin extracellular domains triggered by cytoplasmic signaling pathways after cellular activation.Several alternative models for conformational change have been proposed. Crystal structures obtained of the extracellular domain of ␣V␤3 in Ca 2ϩ (2) or by subsequent soaking in Mn 2ϩ and a cyclic Arg-Gly-Asp (RGD) peptide (3) revealed an unexpected bent conformation, in which the headpiece is folded over and makes extensive contacts with the tailpiece (Fig. 1A). Upon ligand binding, no change was seen at the interface between the I-like and hybrid domains, and only a slight rotation between the ␤-propeller and I-like domains and small movements within the I-like domain were seen. It was hypothesized that both the liganded and unliganded bent structures were in the active state (2, 3). In contrast, NMR, negative stain electron microscopy (EM) with image averaging, ligand binding, physicochemical, and mutational studies show that the bent conformation is physiologic and has low ligand-binding affinity and that activation results in a switchblade-like opening to an extended conformation (4, 5). Two extended conformers were seen that differed...

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Section: Discussionsupporting

confidence: 76%

Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand

Luo

Springer

Takagi

2003

Proc. Natl. Acad. Sci. U.S.A.

146

186

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“…The enhanced avidity gained by the formation of integrin nanoclusters in the cell membrane may possibly serve to enhance cell adhesion by forming nucleation sites for the adhesion (15). In the case of solubilized integrins, the structural biology has received less attention, but important contributions were made by Hantgan et al (17,18). By applying electron microscopy and ultracentrifugation, their work showed that the platelet receptor (integrin a IIb b 3 ) forms stable complexes comprising two or more heterodimers (17), apparently in a process influenced by conformational changes in the ecto domain (18).…”

mentioning

confidence: 97%

“…In the case of solubilized integrins, the structural biology has received less attention, but important contributions were made by Hantgan et al (17,18). By applying electron microscopy and ultracentrifugation, their work showed that the platelet receptor (integrin a IIb b 3 ) forms stable complexes comprising two or more heterodimers (17), apparently in a process influenced by conformational changes in the ecto domain (18). However, no previous studies have addressed the contribution of integrin oligomerization to the function of solubilized receptors and the question of whether integrins other than a IIb b 3 form such complexes.…”

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confidence: 99%

Shedding of Large Functionally Active CD11/CD18 Integrin Complexes from Leukocyte Membranes during Synovial Inflammation Distinguishes Three Types of Arthritis through Differential Epitope Exposure

Gjelstrup

Boesen

Kragstrup

et al. 2010

The Journal of Immunology

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CD18 integrins are adhesion molecules expressed on the cell surface of leukocytes and play a central role in the molecular mechanisms supporting leukocyte migration to zones of inflammation. Recently, it was discovered that CD11a/CD18 is shed from the leukocyte surface in models of inflammation. In this study, we show that shedding of human CD11/CD18 complexes is a part of synovial inflammation in rheumatoid arthritis and spondyloarthritis but not in osteoarthritis. In vivo and in vitro data suggest that the shedding is driven by TNF-α, which links the process to central events in the inflammatory response. The shed complexes contain multiple heterodimers of CD11/CD18, are variable in size, and differ according to the type of synovial inflammation. Furthermore, the differential structures determine the avidity of binding of the complexes to the ICAM-1. With the estimated concentrations of CD11/CD18 in plasma and synovial fluid a significant coverage of binding sites in ICAM-1 for CD18 integrins is expected. Based on cell adhesion experiments in vitro, we hypothesize that the large soluble complexes of CD11/CD18 act in vivo to buffer leukocyte adhesion by competing with the membrane-bound receptors for ICAM-1 binding sites. As reported here for synovial inflammation changes in the concentration or structure of these complexes should be considered as likely contributors to disease activity.

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“…6B. However, further oligomerization may be promoted by fibrinogen/fibrinogen interactions (53) and post-ligand binding changes in ␣ IIb ␤ 3 that expose or re-orient dimerization interfaces (51,52). Recent progress in the structural analysis of ␤ 3 integrins provides a starting point for identifying changes in ␣ IIb ␤ 3 that may affect clustering (11,12,54).…”

Section: Figmentioning

confidence: 99%

Detection of Integrin αIIbβ3Clustering in Living Cells

Buensuceso

Virgilio

Shattil

2003

Journal of Biological Chemistry

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In platelets, bidirectional signaling across integrin ␣ IIb ␤ 3 regulates fibrinogen binding, cytoskeletal reorganization, cell aggregation, and spreading. Because these responses may be influenced by the clustering of ␣ IIb ␤ 3 heterodimers into larger oligomers, we established two independent methods to detect integrin clustering and evaluate factors that regulate this process. In the first, weakly complementing ␤-galactosidase mutants were fused to the C terminus of individual ␣ IIb subunits, and the chimeras were stably expressed with ␤ 3 in Chinese hamster ovary cells. Clustering of ␣ IIb ␤ 3 should bring the mutants into proximity and reconstitute ␤-galactosidase activity. In the second method, ␣ IIb was fused to either a green fluorescent protein (GFP) or Renilla luciferase and transiently expressed with ␤ 3 . Here, integrin clustering should stimulate bioluminescence resonance energy transfer between a cell-permeable luciferase substrate and GFP. These methods successfully detected integrin clustering induced by anti-␣ IIb ␤ 3 antibodies. Significantly, they also detected clustering upon soluble fibrinogen binding to ␣ IIb ␤ 3 . In contrast, no clustering was observed following direct activation of For most cell surface receptors, clustering into dimers or larger oligomers is thought to represent an important regulatory event (1-4). Receptor clustering may be triggered in several ways, for example, through interactions with multivalent ligands, apposition of dimerization interfaces, relief of cytoskeletal constraints, and partitioning into membrane domains such as lipid rafts.Integrins are a family of transmembrane ␣␤ heterodimers that function as cell adhesion and signaling receptors (5). There is substantial indirect evidence that lateral diffusion and clustering influences integrin functions (6, 7). When clustering of recombinant integrins is induced by chemical dimerizers or is abrogated by mutagenesis, their avidity for ligands is affected (8, 9). Indeed, in such systems integrin clustering operates in concert with receptor conformational changes and affinity modulation to determine ligand binding (5, 10 -12). When cells attach to extracellular matrices, integrin clustering promotes the assembly of a range of actin-based complexes, including initial adhesions, focal complexes, and focal adhesions (13-17). These structures, particularly the larger ones, are visible by light microscopy, and they function as signaling centers to help regulate cell motility and gene expression (5, 18). Focal adhesions can be visualized in real time using green fluorescent protein (GFP) 1 chimeras containing focal adhesion targeting sequences (19). On the other hand, smaller nascent integrin clusters containing perhaps a few heterodimers are not visible by routine light microscopy; yet they are likely to function as signaling centers in their own right and represent precursors of the larger adhesion structures (9, 17). In some cases, nascent integrin clusters may recruit functionally important signaling molecules that do...

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Ligand Binding Promotes the Entropy-driven Oligomerization of Integrin αIIbβ3

Cited by 40 publications

References 76 publications

Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand

Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand

Shedding of Large Functionally Active CD11/CD18 Integrin Complexes from Leukocyte Membranes during Synovial Inflammation Distinguishes Three Types of Arthritis through Differential Epitope Exposure

Detection of Integrin αIIbβ3Clustering in Living Cells

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