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: 9.83). 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 04/2014; 310:171-220. DOI:10.1016/B978-0-12-800180-6.00005-0 · 3.42 Impact Factor
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    • "the existence of 3 distinct integrin conformations, with the bent conformation corresponding to the unbound state (Askari et al., 2009). Evidence now indicates that both αvβ3 and α5β1 integrins can also bind ligand in a non-extended, bent conformation (Adair et al., 2005; Askari et al., 2010). The 9EG7 epitope is exposed only upon extension of the β1 integrin subunit to the high affinity state (Askari et al., 2010). "
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    ABSTRACT: Extracellular matrix fibronectin fibrils serve as passive structural supports for the organization of cells into tissues, yet can also actively stimulate a variety of cell and tissue functions, including cell proliferation. Factors that control and coordinate the functional activities of fibronectin fibrils are not known. Here, we compared effects of cell adhesion to vitronectin versus type I collagen on the assembly of and response to, extracellular matrix fibronectin fibrils. The amount of insoluble fibronectin matrix fibrils assembled by fibronectin-null mouse embryonic fibroblasts adherent to collagen- or vitronectin-coated substrates was not significantly different 20 h after fibronectin addition. However, the fibronectin matrix produced by vitronectin-adherent cells was ~ 10-fold less effective at enhancing cell proliferation than that of collagen-adherent cells. Increasing insoluble fibronectin levels with the fibronectin fragment, anastellin did not increase cell proliferation. Rather, native fibronectin fibrils polymerized by collagen- and vitronectin-adherent cells exhibited conformational differences in the growth-promoting, III-1 region of fibronectin, with collagen-adherent cells producing fibronectin fibrils in a more extended conformation. Fibronectin matrix assembly on either substrate was mediated by α5β1 integrins. However, on vitronectin-adherent cells, α5β1 integrins functioned in a lower activation state, characterized by reduced 9EG7 binding and decreased talin association. The inhibitory effect of vitronectin on fibronectin-mediated cell proliferation was localized to the cell-binding domain, but was not a general property of αvβ3 integrin-binding substrates. These data suggest that adhesion to vitronectin allows for the uncoupling of fibronectin fibril formation from downstream signaling events by reducing α5β1 integrin activation and fibronectin fibril extension.
    Matrix biology: journal of the International Society for Matrix Biology 02/2014; 34:33. DOI:10.1016/j.matbio.2014.01.017 · 5.07 Impact Factor
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    • "One of the most important uses of trypsins is to re-suspend cells adherent to the cell culture dish wall during the process of harvesting cells in animal cell cultures [11]. Trypsin is used with EDTA to cleave Ca2+- and Mg2+-dependent integrins bonding the cultured cells to the dish, so that the cells can be suspended in fresh solution and transferred to fresh dishes [11–14]. Since trypsin activity and stability is Ca2+ dependent, the use of EDTA makes it necessary to increase the amount of enzyme and this uncontrolled treatment could alter the physiology, protein expression and metabolism of cultured cells [15–17]. "
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    ABSTRACT: Pig trypsin is routinely used as a biotechnological tool, due to its high specificity and ability to be stored as an inactive stable zymogen. However, it is not an optimum enzyme for conditions found in wound debriding for medical uses and trypsinization processes for protein analysis and animal cell culturing, where low Ca(2+) dependency, high activity in mild conditions and easy inactivation are crucial. We isolated and thermodynamically characterized a highly active cold-adapted trypsin for medical and laboratory use that is four times more active than pig trypsin at 10(°) C and at least 50% more active than pig trypsin up to 50(°) C. Contrary to pig trypsin, this enzyme has a broad optimum pH between 7 and 10 and is very insensitive to Ca(2+) concentration. The enzyme is only distantly related to previously described cryophilic trypsins. We built and studied molecular structure models of this trypsin and performed molecular dynamic calculations. Key residues and structures associated with calcium dependency and cryophilicity were identified. Experiments indicated that the protein is unstable and susceptible to autoproteolysis. Correlating experimental results and structural predictions, we designed mutations to improve the resistance to autoproteolysis and conserve activity for longer periods after activation. One single mutation provided around 25 times more proteolytic stability. Due to its cryophilic nature, this trypsin is easily inactivated by mild denaturation conditions, which is ideal for controlled proteolysis processes without requiring inhibitors or dilution. We clearly show that cold adaptation, Ca(2+) dependency and autolytic stability in trypsins are related phenomena that are linked to shared structural features and evolve in a concerted fashion. Hence, both structurally and evolutionarily they cannot be interpreted and studied separately as previously done.
    PLoS ONE 08/2013; 8(8):e72355. DOI:10.1371/journal.pone.0072355 · 3.23 Impact Factor
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