Three-dimensional EM structure of the ectodomain of integrin αVβ3 in a complex with fibronectin

Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
The Journal of Cell Biology (Impact Factor: 9.69). 04/2005; 168(7):1109-18. DOI: 10.1083/jcb.200410068
Source: PubMed

ABSTRACT Integrins are alphabeta heterodimeric cell surface receptors that mediate transmembrane signaling by binding extracellular and cytoplasmic ligands. The ectodomain of integrin alphaVbeta3 crystallizes in a bent, genuflexed conformation considered to be inactive (unable to bind physiological ligands in solution) unless it is fully extended by activating stimuli. We generated a stable, soluble complex of the Mn(2+)-bound alphaVbeta3 ectodomain with a fragment of fibronectin (FN) containing type III domains 7 to 10 and the EDB domain (FN7-EDB-10). Transmission electron microscopy and single particle image analysis were used to determine the three-dimensional structure of this complex. Most alphaVbeta3 particles, whether unliganded or FN-bound, displayed compact, triangular shapes. A difference map comparing ligand-free and FN-bound alphaVbeta3 revealed density that could accommodate the RGD-containing FN10 in proximity to the ligand-binding site of beta3, with FN9 just adjacent to the synergy site binding region of alphaV. We conclude that the ectodomain of alphaVbeta3 manifests a bent conformation that is capable of stably binding a physiological ligand in solution.

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    • "This model is inadequate to explain the propagation of conformational changes from the cytoplasmic tails to the integrin head. On the other hand, this model assumes ligand binding is possible only when integrins are in an extended, high-affinity conformation, whereas studies have shown soluble avb3 molecules bound to fibronectin maintain their bent conformation (Adair et al., 2005). "
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    ABSTRACT: Cells contain several mechanosensing components that transduce mechanical signals into biochemical cascades. During cell-ECM adhesion, a complex network of molecules mechanically couples the extracellular matrix (ECM), cytoskeleton, and nucleoskeleton. The network comprises transmembrane receptor proteins and focal adhesions, which link the ECM and cytoskeleton. Additionally, recently identified protein complexes extend this linkage to the nucleus by linking the cytoskeleton and the nucleoskeleton. Despite numerous studies in this field, due to the complexity of this network, our knowledge of the mechanisms of cell-ECM adhesion at the molecular level remains remarkably incomplete. Herein, we present a review of the structures of key molecules involved in cell-ECM adhesion, along with an evaluation of their predicted roles in mechanical sensing. Additionally, specific binding events prompted by force-induced conformational changes of each molecule are discussed. Finally, we propose a model for the biomechanical events prominent in cell-ECM adhesion.
    International review of cell and molecular biology 01/2014; 310:171-220. DOI:10.1016/B978-0-12-800180-6.00005-0 · 4.52 Impact Factor
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    • "The 'deadbolt' model proposes that integrin activation and extension occur after ligand binding, whereas the 'switchblade' model suggests that ligand binding only occurs once the integrin heterodimer is in the active extended conformation (Takagi et al. 2001; Beglova et al. 2002; Shimaoka et al. 2002). Debate is still ongoing on which model is correct although the 'switchblade' model is the most widely adopted despite examples still existing of integrin activation occurring while in the bent or partially bent conformation (Adair et al. 2005; Arnaout et al. 2007). "
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    ABSTRACT: Integrins are ubiquitously expressed cell surface receptors that play a critical role in regulating the interaction between a cell and its microenvironment to control cell fate. These molecules are regulated either via their expression on the cell surface or through a unique bidirectional signalling mechanism. However, integrins are just the tip of the adhesome iceberg, initiating the assembly of a large range of adaptor and signalling proteins that mediate the structural and signalling functions of integrin. In this review, we summarise the structure of integrins and mechanisms by which integrin activation is controlled. The different adhesion structures formed by integrins are discussed, as well as the mechanical and structural roles integrins play during cell migration. As the function of integrin signalling can be quite varied based on cell type and context, an in depth understanding of these processes will aid our understanding of aberrant adhesion and migration, which is often associated with human pathologies such as cancer.
    Biophysical Reviews 06/2013; 6(2). DOI:10.1007/s12551-013-0124-0
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    • "There is also accumulating evidence to suggest that integrin need not be fully extended to bind ligand. This includes structural (Adair et al., 2005) and biochemical data (Calzada et al., 2002), as well as biophysical fluorescent resonance energy transfer (FRET) measurements that have attempted to measure conformational changes on the cell surface in response to different agonists (Chigaev et al., 2003, 2007; Coutinho et al., 2007). These experiments suggest a level of complexity in integrin conformational changes not revealed by the structural snapshots so far obtained and pose further questions as to exactly how integrin conformation relates to function and how these changes are coupled. "
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    ABSTRACT: Integrins undergo global conformational changes that specify their activation state. Current models portray the inactive receptor in a bent conformation that upon activation converts to a fully extended form in which the integrin subunit leg regions are separated to enable ligand binding and subsequent signaling. To test the applicability of this model in adherent cells, we used a fluorescent resonance energy transfer (FRET)-based approach, in combination with engineered integrin mutants and monoclonal antibody reporters, to image integrin alpha5beta1 conformation. We find that restricting leg separation causes the integrin to adopt a bent conformation that is unable to respond to agonists and mediate cell spreading. By measuring FRET between labeled alpha5beta1 and the cell membrane, we find extended receptors are enriched in focal adhesions compared with adjacent regions of the plasma membrane. These results demonstrate definitely that major quaternary rearrangements of beta1-integrin subunits occur in adherent cells and that conversion from a bent to extended form takes place at focal adhesions.
    The Journal of Cell Biology 03/2010; 188(6):891-903. DOI:10.1083/jcb.200907174 · 9.69 Impact Factor
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