Unique Disulfide Bonds in Epidermal Growth Factor (EGF) Domains of β3 Affect Structure and Function of αIIbβ3 and αvβ3 Integrins in Different Manner

Mor‐Cohen, Ronit; Rosenberg, Nurit; Einav, Yulia; Zelzion, Ehud; Landau, Meytal; Mansour, Wissam; Averbukh, Yulia; Seligsohn, Uri

doi:10.1074/jbc.m111.311043

Cited by 56 publications

(70 citation statements)

References 64 publications

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“…In contrast, mutations that disturb the domain interfaces all constitutively activate integrin by enforcing extension, tail separation or headpiece opening (supplementary material Fig. S5) (Kashiwagi et al, 1999;Ruiz et al, 2001;Butta et al, 2003;Luo et al, 2003;Luo et al, 2009;Vanhoorelbeke et al, 2009;Mor-Cohen et al, 2012). These mutagenesis studies provide compelling evidence for the requirement of long-range conformational change for integrin activation.…”

Section: Discussionmentioning

confidence: 78%

Modulation of integrin activation and signaling by α1/α1′-helix unbending at the junction

Zhang

Liu

Jiang

et al. 2013

Journal of Cell Science

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SummaryHow conformational signals initiated from one end of the integrin are transmitted to the other end remains elusive. At the ligand-binding bI domain, the a1/a19-helix changes from a bent to a straightened a-helical conformation upon integrin headpiece opening. We demonstrated that a conserved glycine at the a1/a19 junction is crucial for maintaining the bent conformation of the a1/a19-helix in the resting state. Mutations that facilitate a1/a19-helix unbending rendered integrin constitutively active; however, mutations that block the a1/a19-helix unbending abolished soluble ligand binding upon either outside or inside stimuli. Such mutations also blocked ligandinduced integrin extension from outside the cell, but had no effect on talin-induced integrin extension from inside the cell. In addition, integrin-mediated cell spreading, F-actin stress fiber and focal adhesion formation, and focal adhesion kinase activation were also defective in these mutant integrins, although the cells still adhered to immobilized ligands at a reduced level. Our data establish the structural role of the a1/a19 junction that allows relaxation of the a1/a19-helix in the resting state and transmission of bidirectional conformational signals by helix unbending upon integrin activation.

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Section: Discussionmentioning

confidence: 78%

Modulation of integrin activation and signaling by α1/α1′-helix unbending at the junction

Zhang

Liu

Jiang

et al. 2013

Journal of Cell Science

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“…These mutations should disrupt and destabilize the domain structure of the respective repeat as has been observed for cysteine substitutions in EGF repeats of other proteins (Suk et al 2004;Mor-Cohen et al 2012) and should increase the intrinsic flexibility of the mutated repeats. Analyses of single cysteine substitutions (disruption of a single disulfide bridge) in fibrillin showed only localized structural effects but did not exclude the possibility of different effects if other disulfide bridges were disrupted (Suk et al 2004).…”

Section: Discussionmentioning

confidence: 99%

“…Since an EGF-like domain is thought to be an independently folding module (Downing et al 1996), we reasoned that, by disrupting disulphide bridges in individual EGF repeats, the intrinsic structure of neighboring repeats should be only mildly affected, if at all (Suk et al 2004;Mor-Cohen et al 2012), and thus allows one to study the relevance of the structural integrity of individual EGF repeats in the context of full-length DLL1. We decided to exchange cysteine residues 4 and 5 of each individual EGF repeat by glycine, an amino acid that displays intrinsically high flexibility, to disrupt two disulfide bridges of each repeat ( Figure 1A).…”

Section: Generation and Characterization Of Cell Lines Expressing Dllmentioning

confidence: 99%

Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1

et al. 2016

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The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.

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“…β-TD contributes to maintain the bent, inactive conformation of α IIb β 3 ; 12 in fact, the loss of its disulfide bonds causes constitutive activation of α IIb β 3 , [13][14][15] but no information is available on its role in outside-in signaling. Recently, a Japanese family carrying a heterozygous ITGB3 c.2134+1G>A transversion leading to the same integrin β 3 deletion and a similar phenotype was reported.…”

Section: 10mentioning

confidence: 99%

“…Whether the remaining cysteines would then disulfidelink to one another, exchange with the two α1-helix-loop disulfides, or remain as free sulfhydryls is unknown. However, previous site-directed mutagenesis of Cys-655 and 663 leading to the loss of disulfide bonds, resulted in constitutive activation of α IIb β 3 , [13][14][15] suggesting that the same mechanism is responsible for the constitutive α IIb β 3 activation associated with del647-686.…”

mentioning

confidence: 99%

Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia

et al. 2015

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Several patients have been reported to have variant dominant forms of Glanzmann thrombasthenia, associated with macrothrombocytopenia and caused by gain-of-function mutations of ITGB3 or ITGA2B leading to reduced surface expression and constitutive activation of integrin α IIb β 3 . The mechanisms leading to a bleeding phenotype of these patients have never been addressed. The aim of this study was to unravel the mechanism by which ITGB3 mutations causing activation of α IIb β 3 lead to platelet dysfunction and macrothrombocytopenia. Using platelets from two patients carrying the β 3 del647-686 mutation and Chinese hamster ovary cells expressing different α IIb β 3 -activating mutations, we showed that reduced surface expression of α IIb β 3 is due to receptor internalization. Moreover, we demonstrated that permanent triggering of α IIb β 3 -mediated outside-in signaling causes an impairment of cytoskeletal reorganization arresting actin turnover at the stage of polymerization. The induction of actin polymerization by jasplakinolide, a natural toxin that promotes actin nucleation and prevents depolymerization of stress fibers, in control platelets produced an impairment of platelet function similar to that of patients with variant forms of dominant Glanzmann thrombasthenia. del647-686β 3 -transduced murine megakaryocytes generated proplatelets with a reduced number of large tips and asymmetric barbell-proplatelets, suggesting that impaired cytoskeletal rearrangement is the cause of macrothrombocytopenia. These data show that impaired cytoskeletal remodeling caused by a constitutively activated α IIb β 3 is the main effector of platelet dysfunction and macrothrombocytopenia, and thus of bleeding, in variant forms of dominant Glanzmann thrombasthenia. Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia

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Unique Disulfide Bonds in Epidermal Growth Factor (EGF) Domains of β3 Affect Structure and Function of αIIbβ3 and αvβ3 Integrins in Different Manner

Cited by 56 publications

References 64 publications

Modulation of integrin activation and signaling by α1/α1′-helix unbending at the junction

Modulation of integrin activation and signaling by α1/α1′-helix unbending at the junction

Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1

Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia

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