The Three-Dimensional Structure of Integrins and their Ligands, and Conformational Regulation of Cell Adhesion

CBR Institute for Biomedical Research, Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Advances in protein chemistry (Impact Factor: 0.75). 02/2004; 68:29-63. DOI: 10.1016/S0065-3233(04)68002-8
Source: PubMed


Integrins are a structurally elaborate family of adhesion molecules that transmit signals bidirectionally across the plasma membrane by undergoing large-scale structural rearrangements. By regulating cell-cell and cell-matrix contacts, integrins participate in a wide-range of biological interactions including development, tissue repair, angiogenesis, inflammation and hemostasis. From a therapeutic standpoint, integrins are probably the most important class of cell adhesion receptors. Structural investigations on integrin-ligand interactions reveal remarkable features in molecular detail. These details include the atomic basis for divalent cation-dependent ligand binding and how conformational signals are propagated long distances from one domain to another between the cytoplasm and the extracellular ligand binding site that regulate affinity for ligand, and conversely, cytosolic signaling pathways.

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    • "All these events strongly depend on physical linking of fibrinogen, integrin αIIbβ3 and cytoplasmic actin filaments into a stable complex which serves for transmission of contractile force from platelet to platelet within aggregates and from platelet to the thrombogenic surface [3]. Despite numerous elegant studies showing the molecular mechanisms of αIIbβ3 conformational switches, interaction with its ligands, signaling molecules and membrane skeleton proteins [4] [5] [6] [7] [8], regulation of the fibrinogen–αIIbβ3–actin cytoskeleton complex formation at the cellular level has not been fully characterized. It was shown that upon activation platelet cytoskeleton undergoes significant rearrangement including severing of existing actin filaments and assembly of new Thrombosis Research xxx (2014) xxx–xxx Abbreviations: ACD, acid-citrate-dextrose; ADP, adenosine diphosphate; ASA, acetylsalicylic acid; FFD, fibrinogen fragment D; LIBS6, ligand-induced binding site 6; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PPP, platelet-poor plasma; PRP, platelet-rich plasma; SD, standard deviation; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; TBS-T, TBS containing 0.1% (v/v) Tween 20; TRAP, thrombin receptor-activating peptide; TXA 2 , thromboxane A 2 . "
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    ABSTRACT: Thrombus formation in the injured vessel wall is a highly complex process involving various blood-born components that go through specific temporal and spatial changes as observed by intravital videomicroscopy. Platelets bind transiently to the developing thrombus and may either become stably incorporated into or disengage from the thrombus. The aim of the present study was to reveal the processes involved in the formation of a stable thrombus. Platelet-rich plasma and washed platelets were studied by the aggregometer. The aggregate stability was challenged by eptifibatide. Platelet Triton-insoluble fraction was prepared and the actin and αIIb content in the cytoskeleton was analyzed by western blot. Maximal actin polymerization is achieved 1min after platelet activation while maximal αIIbβ3-actin cytoskeleton association requires 5 to 10min of activation and fibrinogen-mediated platelet-to-platelet bridging. Thus, actin polymerization is dependent on platelet activation and requires neither αIIbβ3 integrin occupation nor platelet aggregation. Formation of a stable aggregate requires platelet activation for more than 1min, complete increase in actin cytoskeleton fraction and partial association of αIIbβ3 with the actin cytoskeleton. However, direct αIIbβ3 activation is not sufficient for cytoskeleton complex formation. Thus, stable αIIbβ3-fibrinogen interaction, representing stable aggregate, is achieved after more than 1min agonist activation, involving inside-out and outside-in signaling but not after direct integrin activation, involving only outside-in signaling. Formation of a stable fibrinogen-αIIbβ3-actin cytoskeleton complex is the result of the combined effect of platelet stimulation by soluble agonists, activation of αIIbβ3, fibrinogen binding and platelet-to-platelet bridging. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Thrombosis Research 10/2014; 135(1). DOI:10.1016/j.thromres.2014.10.005 · 2.45 Impact Factor
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    • "Integrins are noncovalently associated αβ heterodimeric cell surface receptors that mediate cell-cell and cell-extracellular matrix adhesions, signaling bidirectionally across the plasma membrane. Integrins play important roles in development, immune cell trafficking and responses, and homeostasis [1], [2], [3]. One of the major leukocyte integrins is the lymphocyte function-associated antigen (LFA)-1, which provides the interactions necessary for immunological synapse formation and adhesion to endothelial cells [4]. "
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    ABSTRACT: Intermolecular contacts between integrin LFA-1 (α(L)β(2)) and ICAM-1 derive solely from the integrin α(L) I domain and the first domain (D1) of ICAM-1. This study presents a crystal structure of the engineered complex of the α(L) I domain and ICAM-1 D1. Previously, we engineered the I domain for high affinity by point mutations that were identified by a directed evolution approach. In order to examine α(L) I domain allostery between the C-terminal α7-helix (allosteric site) and the metal-ion dependent adhesion site (active site), we have chosen a high affinity variant without mutations directly influencing either the position of the α7-helix or the active sites. In our crystal, the α(L) I domain was found to have a high affinity conformation to D1 with its α7-helix displaced downward away from the binding interface, recapitulating a current understanding of the allostery in the I domain and its linkage to neighboring domains of integrins in signaling. To enable soluble D1 of ICAM-1 to fold on its own, we also engineered D1 to be functional by mutations, which were found to be those that would convert hydrogen bond networks in the solvent-excluded core into vdW contacts. The backbone structure of the β-sandwich fold and the epitope for I domain binding of the engineered D1 were essentially identical to those of wild-type D1. Most deviations in engineered D1 were found in the loops at the N-terminal region that interacts with human rhinovirus (HRV). Structural deviation found in engineered D1 was overall in agreement with the function of engineered D1 observed previously, i.e., full capacity binding to α(L) I domain but reduced interaction with HRV.
    PLoS ONE 08/2012; 7(8):e44124. DOI:10.1371/journal.pone.0044124 · 3.23 Impact Factor
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    • "The signal transduction occurs mainly through integrins. Integrins interact with ECM and cytoskeleton, in co-operation with other receptors such as growth factor receptors (Yamada et al., 1980; ffrench-Constant and Colognato, 2004; Humphries et al., 2004; Springer and Wang, 2004). Because heparin binds to fibronectin and several other ECM molecules (Yamada et al., 1980; Barkalow and Schwarzbauer, 1991), integrin participation in the up-regulation of HSPG was investigated. "
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    ABSTRACT: Endothelial cells are a source of physiologically important molecules that are synthesized and released to the blood and/or to the subendothelial extracellular matrix such as a heparan sulfate proteoglycan (HSPG) with antithrombotic properties. Previously, we have shown that heparin stimulates the synthesis and modifies the sulfation pattern of this HSPG. Here the molecular mechanisms involved in the up-regulation of HSPG synthesis by heparin in endothelial cells were decoded. The cells were stimulated with heparin and the expression of HSPG and intracellular pathways were evaluated by a combination of methods involving confocal microscopy, flow cytometry, western blotting analyses, and [(35) S]-sulfate metabolically labeling of the cells. We observed that the up-regulation of HSPG synthesis evoked by heparin is dependent on the interaction of heparin with integrin since RGD peptide abolishes the effect. The activation of integrin leads to tyrosine-phosphorylation of focal adhesion-associated proteins such as FAK, Src, and paxillin. In addition, heparin induces ERK1/2 phosphorylation and inhibitors of Ras and MEK decreased heparin-dependent HSPG synthesis. Moreover, heparin also induced intracellular Ca(2+) release, PLCγ1 (phospholipase Cγ1) and CaMKII (calcium calmodulin kinase II) activation, as well as an increase in nitric oxide (NO) production. Finally, an intracellular Ca(2+) chelator, Ca(2+) signaling inhibitors, and an endothelial NO synthase inhibitor were all able to abolish the effect in heparan sulfate synthesis. In conclusion, the heparin-induced up-regulation of HSPG expression is associated with the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca(2+) /NO pathways. J. Cell. Physiol. © 2011 Wiley-Liss, Inc.
    Journal of Cellular Physiology 06/2012; 227(6). DOI:10.1002/jcp.23018 · 3.84 Impact Factor
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