Identification and characterization of two cation binding sites in the integrin β3 subunit.

Cierniewska-Cieslak, Aleksandra; Cierniewski, Czesław S.; Błędzka, Kamila; Papierak, Malgorzata; Michalec, Lidia; Zhang, Li; Haas, Thomas A.; Plow, Edward F.

doi:10.1074/jbc.a109.112388

Cited by 6 publications

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“…Previous work has identified three I-like MIDAS domain residues critical for ECM binding and/or downstream conformational changes (Bajt and Loftus, 1994). Two residues, D192/S194, were mutated to Alanine, as in vitro studies show that equivalent mutations b3 (D119/S121) and in b1 (D130/S132) abolished binding to ECM ligands (Takada et al, 1992;Bajt and Loftus, 1994;Cierniewska-Cieslak et al, 2002;Chen et al, 2006b). Based on vertebrate studies of Fig.…”

Section: Mutational Analysis Of Drosophila B-integrinmentioning

confidence: 99%

Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis

Pines

Fairchild

Tanentzapf

2010

Developmental Dynamics

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Cell adhesion must be precisely regulated to enable both dynamic morphogenetic processes and the subsequent transition to stable tissue maintenance. Integrins link the intracellular cytoskeleton and extracellular matrix, relaying bidirectional signals across the plasma membrane. In vitro studies have demonstrated that multiple mechanisms control integrin-mediated adhesion; however, their roles during development are poorly understood. We used mutations that activate or deactivate specific functions of vertebrate b-integrins in vitro to investigate how perturbing Drosophila bPS-integrin regulation in developing embryos affects tissue morphogenesis and maintenance. We found that morphogenetic processes use various b-integrin regulatory mechanisms to differing degrees and that conformational changes associated with outside-in activation are essential for developmental integrin functions. Long-term adhesion is also sensitive to integrin dysregulation, suggesting integrins must be continuously regulated to support stable tissue maintenance. Altogether, in vivo phenotypic analyses allowed us to identify the importance of various b-integrin regulatory mechanisms during different morphogenetic processes. Developmental Dynamics 240:36-51,

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Section: Mutational Analysis Of Drosophila B-integrinmentioning

confidence: 99%

Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis

Pines

Fairchild

Tanentzapf

2010

Developmental Dynamics

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“…The LIMBS has been implicated as a positive regulator of ligand or ligand‐mimetic antibody binding, but with variable effects in different integrins 5, 7–11, 13–18. In the integrin β3 subunit, which is common to both αVβ3 and αIIβ3, the residues involved in coordinating its metal ion are D158 (carboxyl oxygen), N215 (amide oxygen), D217 (carboxyl and carbonyl oxygens), P219 (carbonyl oxygen), and E220 (carboxyl oxygen); the latter also coordinates the MIDAS via its other carboxyl oxygen 5, 8.…”

Section: Introductionmentioning

confidence: 99%

Functional and computational studies of the ligand‐associated metal binding site of β3 integrins

Murcia

Jiroušková

et al. 2008

Proteins

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A combination of experimental and computational approaches was used to provide a structural context for the role of the beta3 integrin subunit ligand-associated metal binding site (LIMBS) in the binding of physiological ligands to beta3 integrins. Specifically, we have carried out (1) adhesion assays on cells expressing normal alphaIIbeta3, normal alphaVbeta3, or the corresponding beta3 D217A LIMBS mutants; and (2) equilibrium and nonequilibrium (steered) molecular dynamics (MD) simulations of eptifibatide in complex with either a fully hydrated normal alphaIIbeta3 integrin fragment (alphaIIb beta-propeller and the beta3 betaA (I-like), hybrid, and PSI domains) or the equivalent beta3 D217A mutant. Normal alphaIIbeta3 expressing cells adhered to immobilized fibrinogen and echistatin, whereas cells expressing the alphaIIbeta3 D217A LIMBS mutant failed to adhere to either ligand. Similarly, the equivalent alphaVbeta3 mutant was unable to support adhesion to vitronectin or fibrinogen. The alphaIIbeta3 D217A mutation increased the binding of mAb AP5, which recognizes a ligand-induced binding site (LIBS) in the beta3 PSI domain, indicating that this mutation induced allosteric changes in the protein. Steered MD simulating the unbinding of eptifibatide from either normal alphaIIbeta3 or the equivalent beta3 D217A mutant suggested that the reduction in ligand binding caused by the LIMBS mutant required the loss of both the LIMBS and the metal ion-dependent adhesion site (MIDAS) metal ions. Our computational results indicate that the LIMBS plays a crucial role in ligand binding to alphaIIbeta3 by virtue of its effects on the coordination of the MIDAS.

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“…Thus, induced maturation of α V β 3 integrin affinity stimulates focal adhesion and actin stress fiber formation, as well as promoting recruitment of high‐affinity α V β 3 to focal adhesions. Interestingly, none of these reactions seems to be connected with occupation of MIDAS, ADMIDAS and LIMBS by Mn 2+ , and there is no evidence showing that binding to these sites mediates its stimulatory effect on integrins [6,7,16]. Thus, up to now, the basis for the stimulating effects of Mn 2+ on the ligand‐binding functions of the β 3 integrins is unresolved.…”

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confidence: 99%

Interaction and functional association of protein disulfide isomerase with α_Vβ₃ integrin on endothelial cells

et al. 2008

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Adhesive properties of endothelial cells are influenced by the thioldisulfide balance. However, the molecular mechanism of this effect is unclear, although recent observations indicate that integrin receptors may be direct targets for redox modulation. The purpose of this study was to examine whether protein disulfide isomerase (PDI) is directly involved in this process. As manganese ions are known to affect the thioldisulfide balance and activate integrins to maximal affinity, we searched for PDI interactions with integrins, particularly with αVβ3, in Mn2+‐treated endothelial cells. By employing confocal microscopy, flow cytometry and coimmunoprecipitation experiments, we showed that exposure of endothelial cells to Mn2+ resulted in: (a) the appearance of surface protein thiol groups, which can be found in PDI and αVβ3, and both proteins colocalizing on the cellular surface; and (b) the formation of the PDI–αVβ3 complex, which dissociates upon reduction. In addition, PDI in a complex with αVβ3 induces conversion of the integrin to the ligand‐competent high‐affinity state, as evidenced by increased binding of vitronectin. The membrane‐impermeable sulfhydryl blockers 3‐N‐maleimidylpropionyl biocytin 3‐N‐maleimidylpropionyl biocytin and p‐chloromercuriphenyl sulfonate, as well as the PDI inhibitors bacitracin, MA3 018, and MA3 019, abolished the binding of vitronectin and LM609 to endothelial cells that is activated by Mn2+. Consistently, LM609 almost completely blocked binding of vitronectin to such cells. The formation of the PDI–αVβ3 stoichiometric complex was further demonstrated by surface plasmon resonance analysis, which showed that the initial reversible binding of PDI becomes irreversible in the presence of Mn2+, probably mediated by disulfide bonds. Thus, we show that Mn2+ simultaneously modulates the thiol isomerase activity of PDI that is bound to αVβ3 and induces its transition to the ligand‐competent state, suggesting an alternative mechanism of integrin regulation.

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Identification and characterization of two cation binding sites in the integrin β3 subunit.

Cited by 6 publications

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Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis

Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis

Functional and computational studies of the ligand‐associated metal binding site of β3 integrins

Interaction and functional association of protein disulfide isomerase with α_Vβ₃ integrin on endothelial cells

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Identification and characterization of two cation binding sites in the integrin β3 subunit.

Cited by 6 publications

References 0 publications

Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis

Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis

Functional and computational studies of the ligand‐associated metal binding site of β3 integrins

Interaction and functional association of protein disulfide isomerase with αVβ3 integrin on endothelial cells

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Interaction and functional association of protein disulfide isomerase with α_Vβ₃ integrin on endothelial cells