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Journal articles on the topic 'Integrin affinity'
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1
Hughes, Paul E., and Martin Pfaff. "Integrin affinity modulation." Trends in Cell Biology 8, no. 9 (September 1998): 359–64. http://dx.doi.org/10.1016/s0962-8924(98)01339-7.
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Li, Jing, and Timothy A. Springer. "Energy landscape differences among integrins establish the framework for understanding activation." Journal of Cell Biology 217, no. 1 (November 9, 2017): 397–412. http://dx.doi.org/10.1083/jcb.201701169.
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Why do integrins differ in basal activity, and how does affinity for soluble ligand correlate with cellular adhesiveness? We show that basal conformational equilibrium set points for integrin α4β1 are cell type specific and differ from integrin α5β1 when the two integrins are coexpressed on the same cell. Although α4β1 is easier to activate, its high-affinity state binds vascular cell adhesion molecule and fibronectin 100- to 1,000-fold more weakly than α5β1 binds fibronectin. Furthermore, the difference in affinity between the high- and low-affinity states is more compressed in α4β1 (600- to 800-fold) than in α5β1 (4,000- to 6,000-fold). α4β1 basal conformational equilibria differ among three cell types, define affinity for soluble ligand and readiness for priming, and may reflect differences in interactions with intracellular adaptors but do not predict cellular adhesiveness for immobilized ligand. The measurements here provide a necessary framework for understanding integrin activation in intact cells, including activation of integrin adhesiveness by application of tensile force by the cytoskeleton, across ligand–integrin–adaptor complexes.
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Liddington, R. C., and M. H. Ginsberg. "Integrin activation takes shape." Journal of Cell Biology 158, no. 5 (September 2, 2002): 833–39. http://dx.doi.org/10.1083/jcb.200206011.
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Integrins are cell surface adhesion receptors that are essential for the development and function of multicellular animals. Here we summarize recent findings on the regulation of integrin affinity for ligand (activation), one mechanism by which cells modulate integrin function. The focus is on the structural basis of integrin activation, the role of the cytoplasmic domain in integrin affinity regulation, and potential mechanisms by which activation signals are propagated from integrin cytoplasmic domains to the extracellular ligand-binding domain.
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Dormond, Olivier, Lionel Ponsonnet, Meriem Hasmim, Alessandro Foletti, and Curzio Rüegg. "Manganese-induced integrin affinity maturation promotes recruitment of αVβ3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src." Thrombosis and Haemostasis 92, no. 07 (2004): 151–61. http://dx.doi.org/10.1160/th03-11-0728.
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SummaryIntegrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl2 to increase integrin affinity and monitored clustering of β1 and β3 integrins. In unstimulated HUVEC, β1 integrins were present in fibrillar adhesions, while αVβ3 was detected in peripheral focal adhesions. Clustered β1 and β3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl2-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of β3 high affinity/LIBS epitopes at focal adhesions. MnCl2-induced αVβ3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two parmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced αVβ3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl2 did not alter β1 integrin distribution or β1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl2-induced αVβ3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity αVβ3 to focal adhesions. Affinity-modulated αVβ3 clustering requires PI3-K signaling and is negatively regulate by Src.
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Yu, Tao, Xing Wu, Kiran B. Gupta, and Dennis F. Kucik. "Affinity, lateral mobility, and clustering contribute independently to β2-integrin-mediated adhesion." American Journal of Physiology-Cell Physiology 299, no. 2 (August 2010): C399—C410. http://dx.doi.org/10.1152/ajpcell.00039.2009.
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Affinity changes and avidity modulation both contribute to activation of β2-integrin-mediated adhesion, an essential, early step in inflammation. Avidity modulation, defined as an increase in adhesiveness independent of integrin conformational changes, might be due to integrin clustering, motion, or both. Increased integrin diffusion upon leukocyte activation has been demonstrated, but whether it is proadhesive in itself, or just constitutes a mechanism for integrin clustering, remains unclear. To understand the proadhesive effects of integrin affinity changes, clustering, and motion, an experimental system was devised to separate them. Clustering and integrin motion together were induced by cytochalasin D (CD) without inducing high-affinity; integrin motion could then be frozen by fixation; and high affinity was induced independently by Mn2+. Adhesion was equivalent for fixed and unfixed cells except following pretreatment with CD or Mn2+, which increased adhesion for both. However, fixed cells were less adhesive than unfixed cells after CD, even though integrin clustering was similar. A simple explanation is that CD induces both clustering and integrin motion, fixation then stops motion on fixed cells, but integrins continue to diffuse on unfixed cells, increasing the kinetics of integrin/ICAM-1 interactions to enhance adhesion. Affinity changes are then independent of, and additive to, avidity effects.
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Helsten, Teresa L., Thomas A. Bunch, Hisashi Kato, Jun Yamanouchi, Sharon H. Choi, Alison L. Jannuzi, Chloe C. Féral, Mark H. Ginsberg, Danny L. Brower, and Sanford J. Shattil. "Differences in Regulation ofDrosophilaand Vertebrate Integrin Affinity by Talin." Molecular Biology of the Cell 19, no. 8 (August 2008): 3589–98. http://dx.doi.org/10.1091/mbc.e08-01-0085.
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Integrin-mediated cell adhesion is essential for development of multicellular organisms. In worms, flies, and vertebrates, talin forms a physical link between integrin cytoplasmic domains and the actin cytoskeleton. Loss of either integrins or talin leads to similar phenotypes. In vertebrates, talin is also a key regulator of integrin affinity. We used a ligand-mimetic Fab fragment, TWOW-1, to assess talin's role in regulating Drosophila αPS2βPS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting αPS2βPS affinity is an active process as it is for vertebrate integrins. In contrast to vertebrate integrins, neither talin knockdown by RNA interference nor talin head overexpression had a significant effect on TWOW-1 binding. Furthermore, replacement of the transmembrane or talin-binding cytoplasmic domains of αPS2βPS with those of human αIIbβ3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of αPS2βPS with those of αIIbβ3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type αIIbβ3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talin's integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila αPS2βPS affinity because of structural features inherent in the αPS2βPS extracellular and/or transmembrane domains.
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Tozer, Eileen Collins, Paul E. Hughes, and Joseph C. Loftus. "Ligand binding and affinity modulation of integrins." Biochemistry and Cell Biology 74, no. 6 (December 1, 1996): 785–98. http://dx.doi.org/10.1139/o96-085.
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Integrins are cell adhesion receptors that mediate cell–cell and cell–extracellular matrix interactions. The extracellular domains of these receptors possess binding sites for a diverse range of protein ligands. Ligand binding is divalent cation dependent and involves well-defined motifs in the ligand. Integrins can dynamically regulate their affinity for ligands (inside-out signaling). This ability to rapidly modulate their affinity state is key to their involvement in such processes as cell migration and platelet aggregation. This review will focus on two aspects of integrin function: first, on the molecular basis of ligand–integrin interactions and, second, on the underlying mechanisms controlling the affinity state of integrins for their ligands.Key words: integrins, ligand binding, affinity modulation.
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Dong, Xianchi, Bo Zhao, Fu-Yang Lin, Chafen Lu, Bruce N. Rogers, and Timothy A. Springer. "High integrin αVβ6 affinity reached by hybrid domain deletion slows ligand-binding on-rate." Proceedings of the National Academy of Sciences 115, no. 7 (January 29, 2018): E1429—E1436. http://dx.doi.org/10.1073/pnas.1718662115.
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The role of the hybrid domain in integrin affinity regulation is unknown, as is whether the kinetics of ligand binding is modulated by integrin affinity state. Here, we compare cell surface and soluble integrin αVβ6 truncation mutants for ligand-binding affinity, kinetics, and thermodynamics. Removal of the integrin transmembrane/cytoplasmic domains or lower legs has little effect on αVβ6 affinity, in contrast to β1 integrins. In integrin opening, rearrangement at the interface between the βI and hybrid domains is linked to remodeling at the ligand-binding site at the opposite end of the βI domain, which greatly increases in affinity in the open conformation. The larger size of the βI-hybrid interface in the closed state suggests that the hybrid domain stabilizes closing. In agreement, deletion of the hybrid domain raised affinity by 50-fold. Surface plasmon resonance and isothermal titration calorimetry gave similar results and the latter revealed tradeoffs between enthalpy and entropy not apparent from affinity. At extremely high affinity reached in Mn2+ with hybrid domain truncation, αVβ6 on-rate for both pro-TGF-β1 and fibronectin declined. The results suggest that the open conformation of αVβ6 has lower on-rate than the closed conformation, correlate with constriction of the ligand-binding pocket in open αVβ6 structures, and suggest that the extended-closed conformation is kinetically selected for ligand binding. Subsequent transition to the extended-open conformation is stabilized by its much higher affinity for ligand and would also be stabilized by force exerted across ligand-bound integrins by the actin cytoskeleton.
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Bialkowska, Katarzyna, Jun Qin, and Edward F. Plow. "Phosphorylation of Kindlins and the Control of Integrin Function." Cells 10, no. 4 (April 7, 2021): 825. http://dx.doi.org/10.3390/cells10040825.
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Integrins serve as conduits for the transmission of information between cells and their extracellular environment. Signaling across integrins is bidirectional, transducing both inside-out and outside-signaling. Integrin activation, a transition from a low affinity/avidity state to a high affinity/avidity state for cognate ligands, is an outcome of inside-signaling. Such activation is particularly important for the recognition of soluble ligands by blood cells but also influences cell-cell and cell-matrix interactions. Integrin activation depends on a complex series of interactions, which both accelerate and inhibit their interconversion from the low to the high affinity/avidity state. There are three components regarded as being most proximately involved in integrin activation: the integrin cytoplasmic tails, talins and kindlins. The participation of each of these molecules in integrin activation is highly regulated by post-translation modifications. The importance of targeted phosphorylation of integrin cytoplasmic tails and talins in integrin activation is well-established, but much less is known about the role of post-translational modification of kindlins. The kindlins, a three-member family of 4.1-ezrin-radixin-moesin (FERM)-domain proteins in mammals, bind directly to the cytoplasmic tails of integrin beta subunits. This commentary provides a synopsis of the emerging evidence for the role of kindlin phosphorylation in integrin regulation.
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Mahabeleshwar, Ganapati H., Juhua Chen, Weiyi Feng, Payaningal R. Somanath, Olga V. Razorenova, and Tatiana V. Byzova. "Integrin affinity modulation in angiogenesis." Cell Cycle 7, no. 3 (February 2008): 335–47. http://dx.doi.org/10.4161/cc.7.3.5234.
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Sun, Guangyu, Emilie Guillon, and Scott A. Holley. "Integrin intra-heterodimer affinity inversely correlates with integrin activatability." Cell Reports 35, no. 10 (June 2021): 109230. http://dx.doi.org/10.1016/j.celrep.2021.109230.
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Woodside, D. G., S. Liu, and M. H. Ginsberg. "Integrin Activation." Thrombosis and Haemostasis 86, no. 07 (2001): 316–23. http://dx.doi.org/10.1055/s-0037-1616229.
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SummaryIntegrins are cell surface adhesion receptors that participate in a variety of important processes throughout the vasculature. Here we summarize some recent findings on the regulation of integrin mediated cellular adhesion. Particular emphasis is placed on the regulation of integrin affinity for ligand (activation), although this is just one mechanism by which regulation of integrin-dependent cell adhesion can occur. Also discussed are recent observations on the structural basis of integrin activation, the role of the cytoplasmic domain in integrin affinity regulation, and potential mechanisms by which activation signals are propagated from integrin cytoplasmic domains to the extracellular ligand binding domain.
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Kinashi, Tatsuo, Tetsuo Asaoka, Ruri Setoguchi, and Kiyoshi Takatsu. "Affinity Modulation of Very Late Antigen-5 Through Phosphatidylinositol 3-Kinase in Mast Cells." Journal of Immunology 162, no. 5 (March 1, 1999): 2850–57. http://dx.doi.org/10.4049/jimmunol.162.5.2850.
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Abstract Adhesiveness of integrins is up-regulated rapidly by a number of molecules, including growth factors, cytokines, chemokines, and other cell surface receptors, through a mechanism termed inside-out signaling. The inside-out signaling pathways are thought to alter integrin affinity for ligand, or cell surface distribution of integrin by diffusion/clustering. However, it remains to be clarified whether any physiologically relevant agonists induce a rapid change in the affinity of β1 integrins and how ligand-binding affinity is modulated upon stimulation. In this study, we reported that affinity of β1 integrin very late Ag-5 (VLA-5) for fibronectin was rapidly increased in bone marrow-derived mast cells by Ag cross-linking of FcεRI. Ligand-binding affinity of VLA-5 was also augmented by receptor tyrosine kinases when the phospholipase Cγ-1/protein kinase C pathway was inhibited. Wortmannin suppressed induction of the high affinity state VLA-5 in either case. Conversely, introduction of a constitutively active p110 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) increased the binding affinity for fibronectin. Failure of a constitutively active Akt to stimulate adhesion suggested that the affinity modulation mechanisms mediated by PI 3-kinase are distinct from the mechanisms to control growth and apoptosis by PI 3-kinase. Taken together, our findings demonstrated that the increase of affinity of VLA-5 was induced by physiologically relevant stimuli and PI 3-kinase was a critical affinity modulator of VLA-5.
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Pinon, Perrine, Jenita Pärssinen, Patricia Vazquez, Michael Bachmann, Rolle Rahikainen, Marie-Claude Jacquier, Latifeh Azizi, et al. "Talin-bound NPLY motif recruits integrin-signaling adapters to regulate cell spreading and mechanosensing." Journal of Cell Biology 205, no. 2 (April 28, 2014): 265–81. http://dx.doi.org/10.1083/jcb.201308136.
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Integrin-dependent cell adhesion and spreading are critical for morphogenesis, tissue regeneration, and immune defense but also tumor growth. However, the mechanisms that induce integrin-mediated cell spreading and provide mechanosensing on different extracellular matrix conditions are not fully understood. By expressing β3-GFP-integrins with enhanced talin-binding affinity, we experimentally uncoupled integrin activation, clustering, and substrate binding from its function in cell spreading. Mutational analysis revealed Tyr747, located in the first cytoplasmic NPLY747 motif, to induce spreading and paxillin adapter recruitment to substrate- and talin-bound integrins. In addition, integrin-mediated spreading, but not focal adhesion localization, was affected by mutating adjacent sequence motifs known to be involved in kindlin binding. On soft, spreading-repellent fibronectin substrates, high-affinity talin-binding integrins formed adhesions, but normal spreading was only possible with integrins competent to recruit the signaling adapter protein paxillin. This proposes that integrin-dependent cell–matrix adhesion and cell spreading are independently controlled, offering new therapeutic strategies to modify cell behavior in normal and pathological conditions.
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Sethi, Tariq, Mark H. Ginsberg, Julian Downward, and Paul E. Hughes. "The Small GTP-binding Protein R-Ras Can Influence Integrin Activation by Antagonizing a Ras/Raf-initiated Integrin Suppression Pathway." Molecular Biology of the Cell 10, no. 6 (June 1999): 1799–809. http://dx.doi.org/10.1091/mbc.10.6.1799.
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The rapid modulation of ligand-binding affinity (“activation”) is a central property of the integrin family of cell adhesion receptors. The small GTP-binding protein Ras and its downstream effector kinase Raf-1 suppress integrin activation. In this study we explored the relationship between Ras and the closely related small GTP-binding protein R-Ras in modulating the integrin affinity state. We found that R-Ras does not seem to be a direct activator of integrins in Chinese hamster ovary cells. However, we observed that GTP-bound R-Ras strongly antagonizes the Ras/Raf-initiated integrin suppression pathway. Furthermore, this reversal of the Ras/Raf suppressor pathway does not seem to be via a competition between Ras and R-Ras for common downstream effectors or via an inhibition of Ras/Raf-induced MAP kinase activation. Thus, R-Ras and Ras may act in concert to regulate integrin affinity via the activation of distinct downstream effectors.
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Huttenlocher, A., M. H. Ginsberg, and A. F. Horwitz. "Modulation of cell migration by integrin-mediated cytoskeletal linkages and ligand-binding affinity." Journal of Cell Biology 134, no. 6 (September 15, 1996): 1551–62. http://dx.doi.org/10.1083/jcb.134.6.1551.
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Integrin cell surface adhesion receptors play a central role in mediating cell migration. We have developed a model system consisting of CHO cells ectopically expressing the alpha IIb beta 3 integrin to study integrin affinity and cytoskeletal interactions during cell migration. The alpha IIb beta 3 integrins are suited for study of integrin receptors during cell migration because they are well characterized with respect to ligand binding, cytoskeletal interactions, and signal transduction, and mutants with altered receptor function are available. The alpha IIb beta 3 receptor specifically mediates migration of alpha IIb beta 3-transfected CHO cells. The migration of transfected CHO cells was studied on a fibrinogen substrate both by time lapse videomicroscopy and by random and haptotactic transwell assays. Haptotactic and random transwell assays measured distinct aspects of migration, with the random transwell assay correlating most closely with time lapse videomicroscopy. Mutations in the cytoplasmic domains that increase ligand affinity or activation of the alpha IIb beta 3 receptor into a high affinity state by the LIBS6 antibody decreased the migration rate. Likewise, mutations that increase cytoskeletal organization without affecting affinity also decreased the migration rate. In contrast, truncation of the beta chain, which alters cytoskeletal associations as assayed by absence of focal adhesions, decreased haptotactic migration while increasing random migration. These effects on the migration rate were partially compensated for by altering substrate concentration, demonstrating optimum substrate concentrations that supported maximal migration. For example, cells expressing integrins locked in the high affinity state showed maximal migration at lower substrate concentrations than cells expressing low affinity receptor. Together, these results implicate the strength of adhesion between cell and substrate, as modulated by receptor affinity, organization of adhesive complexes, and substrate concentration, as important regulators of cell migration rate. Further, we demonstrate a dominant effect of high affinity integrin in inhibiting migration regardless of the organization of adhesive complexes. These observations have potential implications for tumor metastasis and its therapy.
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Martin-Bermudo, Maria D., Olga M. Dunin-Borkowski, and Nicholas H. Brown. "Modulation of Integrin Activity is Vital for Morphogenesis." Journal of Cell Biology 141, no. 4 (May 18, 1998): 1073–81. http://dx.doi.org/10.1083/jcb.141.4.1073.
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Cells can vary their adhesive properties by modulating the affinity of integrin receptors. The activation and inactivation of integrins by inside-out mechanisms acting on the cytoplasmic domains of the integrin subunits has been demonstrated in platelets, lymphocytes, and keratinocytes. We show that in the embryo, normal morphogenesis requires the α subunit cytoplasmic domain to control integrin adhesion at the right times and places. PS2 integrin (αPS2βPS) adhesion is normally restricted to the muscle termini, where it is required for attaching the muscles to the ends of other muscles and to specialized epidermal cells. Replacing the wild-type αPS2 with mutant forms containing cytoplasmic domain deletions results in the rescue of the majority of defects associated with the absence of the αPS2 subunit, however, the mutant PS2 integrins are excessively active. Muscles containing these mutant integrins make extra muscle attachments at aberrant positions on the muscle surface, disrupting the muscle pattern and causing embryonic lethality. A gain- of-function phenotype is not observed in the visceral mesoderm, showing that regulation of integrin activity is tissue-specific. These results suggest that the αPS2 subunit cytoplasmic domain is required for inside-out regulation of integrin affinity, as has been seen with the integrin αIIbβ3.
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Kim, Chungho, Tong-Lay Lau, Tobias S. Ulmer, and Mark H. Ginsberg. "Interactions of platelet integrin αΙΙb and β3 transmembrane domains in mammalian cell membranes and their role in integrin activation." Blood 113, no. 19 (May 7, 2009): 4747–53. http://dx.doi.org/10.1182/blood-2008-10-186551.
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Abstract Clustering and occupancy of platelet integrin αIIbβ3 (GPIIb-IIIa) generate biologically important signals: conversely, intracellular signals increase the integrins' affinity, leading to integrin activation; both forms of integrin signaling play important roles in hemostasis and thrombosis. Indirect evidence implicates interactions between integrin α and β transmembrane domains (TMDs) and cytoplasmic domains in integrin signaling; however, efforts to directly identify these associations have met with varying and controversial results. In this study, we develop mini-integrin affinity capture and use it in combination with nuclear magnetic resonance spectroscopy to show preferential heterodimeric association of integrin αIIbβ3 TMD tails via specific TMD interactions in mammalian cell membranes in lipid bicelles. Furthermore, charge reversal mutations at αIIb(R995)β3(D723) confirm a proposed salt bridge and show that it stabilizes the TMD-tail association; talin binding to the β3 tail, which activates the integrin, disrupts this association. These studies establish the preferential heterodimeric interactions of integrin αIIbβ3 TMD tails in mammalian cell membranes and document their role in integrin signaling.
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Palecek, S. P., A. Huttenlocher, A. F. Horwitz, and D. A. Lauffenburger. "Physical and biochemical regulation of integrin release during rear detachment of migrating cells." Journal of Cell Science 111, no. 7 (April 1, 1998): 929–40. http://dx.doi.org/10.1242/jcs.111.7.929.
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Cell migration can be considered as a repeated cycle of membrane protrusion and attachment, cytoskeletal contraction and rear detachment. At intermediate and high levels of cell-substratum adhesiveness, cell speed appears to be rate-limited by rear detachment, specifically by the disruption of cytoskeleton-adhesion receptor-extracellular matrix (ECM) linkages. Often, cytoskeletal linkages fracture to release integrin adhesion receptors from the cell. Cell-extracellular matrix bonds may also dissociate, allowing the integrins to remain with the cell. To investigate molecular mechanisms involved in fracturing these linkages and regulating cell speed, we have developed an experimental system to track integrins during the process of rear retraction in Chinese hamster ovary (CHO) cells. Integrin expression level was varied by transfecting CHO B2 cells, which express very little endogenous alpha5 integrin, with a plasmid containing human alpha5 integrin cDNA and sorting the cells into three populations with different alpha5 expression levels. Receptor/ligand affinity was varied using CHO cells transfected with either alphaIIbbeta3 or alphaIIbbeta3(beta1-2), a high affinity variant. alphaIIbbeta3(beta1-2) is activated to a higher affinity state with an anti-LIBS2 antibody. Fluorescent probes were conjugated to non-adhesion perturbing anti-integrin antibodies, which label integrins in CHO cells migrating on a matrix-coated glass coverslip. The rear retraction area was determined using phase contrast microscopy and integrins initially in this area were tracked by fluorescence microscopy and a cooled CCD camera. We find that rear retraction rate appears to limit cell speed at intermediate and high adhesiveness, but not at low adhesiveness. Upon rear retraction, the amount of integrin released from the cell increases as extracellular matrix concentration, receptor level and receptor-ligand affinity increase. In fact, integrin release is a constant function of cell-substratum adhesiveness and the number of cell-substratum bonds. In the adhesive regime where rear detachment limits the rate of cell migration, cell speed has an inverse relationship to the amount of integrin released at the rear of the cell. At high cell-substratum adhesiveness, calpain, a Ca2+-dependent protease, is also involved in release of cytoskeletal linkages during rear retraction. Inhibition of calpain results in decreased integrin release from the cell membrane, and consequently a decrease in cell speed, during migration. These observations suggest a model for rear retraction in which applied tension and calpain-mediated cytoskeletal linkage cleavage are required at high adhesiveness, but only applied tension is required at low adhesiveness.
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Fan, Zhichao, Sara McArdle, Zbigniew Mikulski, Edgar Gutierrez, Mark Ginsberg, Alex Groisman, and Klaus Ley. "Neutrophil recruitment limited by high affinity bent β2 integrin binding ligand in cis." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 119.5. http://dx.doi.org/10.4049/jimmunol.196.supp.119.5.
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Abstract Neutrophils are essential in innate immunity and inflammation. Many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high affinity conformation with an “open” headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here, by using high-resolution quantitative dynamic foot printing (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, we show that a substantial fraction of β2 integrins on human neutrophils acquires an unexpected E−H+ conformation. Beside the observations consistent with the canonical switchblade model, we discovered a new alternative model that some β2 integrins transition from E−H− to E−H+ to E+H+ during activation. Using flow cytometry based Förster resonance energy transfer (FRET) assay, we find out that E−H+ β2 integrin binds its ligand intercellular adhesion molecule 1 (ICAM-1) in cis. And this cis binding significantly inhibits the conformational transitioning of β2 integrin from E−H+ to E+H+ and the neutrophil adhesion under flow, which indicates a novel endogenous anti-inflammatory mechanism inhibits neutrophil accumulation and inflammation.
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Kolasangiani, Reza, Tamara C. Bidone, and Martin A. Schwartz. "Integrin Conformational Dynamics and Mechanotransduction." Cells 11, no. 22 (November 12, 2022): 3584. http://dx.doi.org/10.3390/cells11223584.
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The function of the integrin family of receptors as central mediators of cell-extracellular matrix (ECM) and cell–cell adhesion requires a remarkable convergence of interactions and influences. Integrins must be anchored to the cytoskeleton and bound to extracellular ligands in order to provide firm adhesion, with force transmission across this linkage conferring tissue integrity. Integrin affinity to ligands is highly regulated by cell signaling pathways, altering affinity constants by 1000-fold or more, via a series of long-range conformational transitions. In this review, we first summarize basic, well-known features of integrin conformational states and then focus on new information concerning the impact of mechanical forces on these states and interstate transitions. We also discuss how these effects may impact mechansensitive cell functions and identify unanswered questions for future studies.
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Wen, Lai, Alex Marki, Zhihao Wang, Marco Orecchioni, Jeffrey Makings, Kenneth Kim, William B. Kiosses, Zbigniew Mikulski, and Klaus Ley. "A new β2 integrin activation reporter mouse reveals localized intra- and extra-vascular neutrophil integrin activation in vivo." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 105.07. http://dx.doi.org/10.4049/jimmunol.208.supp.105.07.
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Abstract β2 integrins (LFA-1, Mac-1, CD11c-CD18, and CD11d-CD18) are leukocyte-specific adhesion receptors that play critical roles in leukocyte recruitment, as well as other immunological processes such as phagocytosis and immunological synapse formation. Adhesion of leukocytes to other cells such as endothelial cells are regulated by integrin affinity changes for their ligands (“activation”). Human β2 integrin activation can be detected by reporter antibodies including mAb24 and KIM127. No such activation epitopes are known in mouse β2 integrins. Because of the lack of mouse β2 integrin activation reporter antibodies, nothing is known about β2 integrin activation in vivo. Here, we generated a humanized β2 integrin knockin mouse by targeting the human β2 integrin coding sequence into the mouse Itgb2 locus. We show that this enables imaging of β2 integrin activation using the KIM127 (extension conformation) and mAb24 (high affinity) reporter antibodies. Human β2 pairs with the mouse integrin α chains, yielding normal expression of the β2 integrins LFA-1, Mac-1 and CD11c-CD18 in all major leukocyte populations. Using a CXCL1-induced acute inflammation model, we uncovered the dynamics and subcellular localization of β2 integrin activation in arresting neutrophils in vivo in venules of the mouse cremaster muscle. Activated integrins in arresting neutrophils in vivo are concentrated at the interface of neutrophils and the endothelium at the rear side of neutrophils facing against the blood flow. In a high-dose lipopolysaccharide (LPS) model, we found that β2 integrins are activated in association with elevated neutrophil adhesion in lung and liver. Thus, these mice, for the first time, enable studies into β2 integrin activation in vivo. This work was supported by grants from the National Institutes of Health, USA (HL078784 to K.L.) and a Postdoctoral Fellowship (19POST34450228 to L.W.) from the American Heart Association, USA.
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Hyduk, Sharon J., Jiwon Oh, Haiyan Xiao, Mian Chen, and Myron I. Cybulsky. "Paxillin selectively associates with constitutive and chemoattractant-induced high-affinity α4β1 integrins: implications for integrin signaling." Blood 104, no. 9 (November 1, 2004): 2818–24. http://dx.doi.org/10.1182/blood-2003-12-4402.
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Abstract Leukocyte α4β1 integrins regulate hematopoietic and lymphoid development, as well as the emigration of circulating cells to sites of inflammation. Because vascular cell adhesion molecule-1 (VCAM-1) binding to high-affinity α4β1 is stable, these integrins can be detected and selectively precipitated from cell lysates using VCAM-1/Fc. With this approach, high-affinity α4β1 integrin expression was demonstrated on lymphocytes in the bone marrow, thymus, spleen, and the peritoneal cavity of normal mice, but not in peripheral lymph nodes. Immature lymphocytes preferentially expressed high-affinity α4β1 in the bone marrow and thymus. Paxillin is a cytoplasmic adaptor molecule that can bind to the α4 tail and initiate signaling. Paxillin was associated selectively with high-affinity integrins that were isolated from human Jurkat T cells or from murine tissues, and blotting with a phospho-specific antibody demonstrated that Ser988 in the α4 cytoplasmic tail was dephosphorylated in high-affinity but not low-affinity integrins. A rapid and transient α4β1 affinity up-regulation in formyl peptide receptor-transfected U937 cells stimulated with N-formyl-methyonyl-leucyl-phenylalanine (fMLP) correlated temporally with induced paxillin binding to α4 integrins. These data suggest that ligand binding to high-affinity α4β1 integrins may initiate outside-in signaling cascades through paxillin that regulate leukocyte maturation and emigration. (Blood. 2004;104:2818-2824)
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Yue, Jiao, YouDong Pan, LiFang Sun, Kun Zhang, Jie Liu, Ling Lu, and JianFeng Chen. "The Unique Disulfide Bond-stabilized W1 β4-β1 Loop in the α4 β-Propeller Domain Regulates Integrin α4β7 Affinity and Signaling." Journal of Biological Chemistry 288, no. 20 (April 3, 2013): 14228–37. http://dx.doi.org/10.1074/jbc.m113.462630.
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Integrin α4β7 mediates rolling and firm adhesion of lymphocytes pre- and post-activation, which is distinct from most integrins only mediating firm cell adhesion upon activation. This two-phase cell adhesion suggests a unique molecular basis for the dynamic interaction of α4β7 with its ligand, mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Here we report that a disulfide bond-stabilized W1 β4-β1 loop in α4 β-propeller domain plays critical roles in regulating integrin α4β7 affinity and signaling. Either breaking the disulfide bond or deleting the disulfide bond-occluded segment in the W1 β4-β1 loop inhibited rolling cell adhesion supported by the low-affinity interaction between MAdCAM-1 and inactive α4β7 but negligibly affected firm cell adhesion supported by the high-affinity interaction between MAdCAM-1 and Mn2+-activated α4β7. Additionally, disrupting the disulfide bond or deleting the disulfide bond-occluded segment not only blocked the conformational change and activation of α4β7 triggered by talin or phorbol-12-myristate-13-acetate via inside-out signaling but also disrupted integrin-mediated outside-in signaling and impaired phosphorylation of focal adhesion kinase and paxillin. Thus, these findings reveal a particular molecular basis for α4β7-mediated rolling cell adhesion and a novel regulatory element of integrin affinity and signaling.
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Jovanović, Jelena, Sarah Iqbal, Sacha Jensen, Helen Mardon, and Penny Handford. "Fibrillin–integrin interactions in health and disease." Biochemical Society Transactions 36, no. 2 (March 20, 2008): 257–62. http://dx.doi.org/10.1042/bst0360257.
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Human fibrillin-1 is the major structural protein of extracellular matrix 10–12 nm microfibrils. It has a disulfide-rich modular organization which consists primarily of cbEGF (Ca2+-binding epidermal growth factor-like) domains and TB (transforming growth factor β-binding protein-like) domains. TB4 contains an RGD (Arg-Gly-Asp) integrin-binding motif. The atomic structure of this region has been solved by X-ray crystallography and shows the TB4 and flanking cbEGF domains to be arranged as a tetragonal pyramid with N- and C-termini exposed at opposite ends of the fragment. The RGD integrin-binding motif is located within a flexible loop. We have used a variety of biophysical, biochemical and cell biology methods to investigate the molecular properties of integrin–fibrillin-1 interactions and have demonstrated that recombinant fibrillin-1 domain fragments mediate binding to integrins αVβ3, α5β1 and αVβ6. Integrin αVβ3 is a high-affinity fibrillin-1 receptor (Kd ∼40 nM), whereas integrins αVβ6 and α5β1 show moderate-affinity (Kd ∼450 nM) and low-affinity (Kd >1 μM) binding respectively. Different patterns of α5β1 distribution are seen when human keratinocytes and fibroblasts are plated on to fibrillin domain fragments compared with those seen for fibronectin, suggesting that fibrillin may cause a lesser degree or different type of intracellular signalling. A number of disease-causing mutations which affect the TB4 domain have been identified. These are being investigated for their effects on integrin binding and/or changes in intramolecular structure.
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Fan, Zhichao, Lai Wen, Yi-Ting Yeh, William B. Kiosses, Edgar Gutierrez, Alex Groisman, Joshua Francois, et al. "Kindlin-3 organizes a ring of clustered high affinity β2 integrins during human neutrophil spreading under flow." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 220.1. http://dx.doi.org/10.4049/jimmunol.204.supp.220.1.
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Abstract Neutrophils are vital for inflammation and immune defense. Dependent on β2 integrins, spherical neutrophils spread on vascular endothelium after arrest, which is critical for their recruitment from circulation to resist high shear flow and to initiate intravascular crawling. Here, we use high-resolution quantitative dynamic footprinting microscopy to monitor neutrophil spreading on a substrate of recombinant ICAM-1 and P-selectin under flow. A homogenous binding assay using the conformation-reporter antibodies mAb24 (reporting high-affinity β2, H+) and KIM127 (reporting extended β2, E+) showed three conformations of activated β2 integrins. E−H+ β2 integrins increased before E+H− and E+H+ conformations at the beginning of neutrophil spreading. Integrin extension depended on Syk-mediated integrin outside-in signaling. The ring of E−H+ and E+H+, but not E+H− β2 integrins was fully formed during late neutrophil spreading just before migration. Using kindlin-3-GFP fusion proteins, a ring of kindlin-3 was observed before the ring of H+ integrins appeared. These findings show spatially coordinated integrin activation during spreading. The previously unrecognized E−H+ conformation is the pioneer integrin for neutrophil spreading and appears to be organized by kindlin-3.
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D’Souza-Schorey, Crislyn, Benjamin Boettner, and Linda Van Aelst. "Rac Regulates Integrin-Mediated Spreading and Increased Adhesion of T Lymphocytes." Molecular and Cellular Biology 18, no. 7 (July 1, 1998): 3936–46. http://dx.doi.org/10.1128/mcb.18.7.3936.
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ABSTRACT Leukocyte adhesion to the extracellular matrix (ECM) is tightly controlled and is vital for the immune response. Circulating lymphocytes leave the bloodstream and adhere to ECM components at sites of inflammation and lymphoid tissues. Mechanisms for regulating T-lymphocyte–ECM adhesion include (i) an alteration in the affinity of cell surface integrin receptors for their extracellular ligands and (ii) an alteration of events following postreceptor occupancy (e.g., cell spreading). Whereas H-Ras and R-Ras were previously shown to affect T-cell adhesion by altering the affinity state of the integrin receptors, no signaling molecule has been identified for the second mechanism. In this study, we demonstrated that expression of an activated mutant of Rac triggered dramatic spreading of T cells and their increased adhesion on immobilized fibronectin in an integrin-dependent manner. This effect was not mimicked by expression of activated mutant forms of Rho, Cdc42, H-Ras, or ARF6, indicating the unique role of Rac in this event. The Rac-induced spreading was accompanied by specific cytoskeletal rearrangements. Also, a clustering of integrins at sites of cell adhesion and at the peripheral edges of spread cells was observed. We demonstrate that expression of RacV12 did not alter the level of expression of cell surface integrins or the affinity state of the integrin receptors. Moreover, our results indicate that Rac plays a role in the regulation of T-cell adhesion by a mechanism involving cell spreading, rather than by altering the level of expression or the affinity of the integrin receptors. Furthermore, we show that the Rac-mediated signaling pathway leading to spreading of T lymphocytes did not require activation of c-Jun kinase, serum response factor, or pp70S6 kinase but appeared to involve a phospholipid kinase.
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O'Toole, TE, Y. Katagiri, RJ Faull, K. Peter, R. Tamura, V. Quaranta, JC Loftus, SJ Shattil, and MH Ginsberg. "Integrin cytoplasmic domains mediate inside-out signal transduction." Journal of Cell Biology 124, no. 6 (March 15, 1994): 1047–59. http://dx.doi.org/10.1083/jcb.124.6.1047.
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We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.
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Schürpf, Thomas, and Timothy A. Springer. "Regulation of integrin affinity on cell surfaces." EMBO Journal 30, no. 23 (September 23, 2011): 4712–27. http://dx.doi.org/10.1038/emboj.2011.333.
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Karsan, A. "Notch and Integrin Affinity: A Sticky Situation." Science Signaling 1, no. 2 (January 8, 2008): pe2. http://dx.doi.org/10.1126/stke.12pe2.
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Rose, David M., Valentin Grabovsky, Ronen Alon, and Mark H. Ginsberg. "The Affinity of Integrin α4β1Governs Lymphocyte Migration." Journal of Immunology 167, no. 5 (September 1, 2001): 2824–30. http://dx.doi.org/10.4049/jimmunol.167.5.2824.
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Brown, Simon B., and Ian Dransfield. "Electric Fields and Inflammation: May the Force be with You." Scientific World JOURNAL 8 (2008): 1280–94. http://dx.doi.org/10.1100/tsw.2008.158.
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Integrins are a family of ubiquitous cell surface receptors comprising heterodimers of β and α chains that are required for cell adhesion and motility. Integrin-dependent adhesion and signaling is associated with major conformational changes in the ectodomain as it shifts from a low-affinity “bent” to a high-affinity “extended” structure. The ability of a cell to regulate dynamically the affinity or activation state of an integrin, and hence its binding to extracellular matrix or cell adhesion molecules, is assumed to be driven by intracellular signaling events transmitted by protein binding to the cytoplasmic tail. The binding of an integrin to its ligand can then transmit signals back into the cell to regulate the formation of a macromolecular focal adhesion complex that effectively anchors the cytoskeleton to the adhesion site. Many proteins have been reported to associate physically and functionally with integrins, leading to altered signaling events. A particularly intriguing molecular association exists between integrins and transmembrane proteins that gate the movement of charge, especially voltage-gated potassium channels, although the significance of this interaction is not understood. Although ample evidence indicates that the engagement of integrins can promote potassium efflux by both excitable and nonexcitable cells, we speculate the converse, that the activation state of integrins is dynamically regulated by changes in a transmembrane potential. In this way, direct-current electric fields generated at a site of tissue injury can promote the galvanotaxis or directed migration of cells involved in tissue repair and inflammation.
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Hyduk, Sharon J., Haiyan Xiao, and Myron I. Cybulsky. "PAXILLIN SELECTIVELY ASSOCIATES WITH HIGH AFFINITY α4β1 INTEGRINS: IMPLICATIONS FOR INTEGRIN SIGNALING." Cardiovascular Pathology 13, no. 3 (May 2004): 6. http://dx.doi.org/10.1016/j.carpath.2004.03.012.
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Craig, David H., Christopher P. Gayer, Keri L. Schaubert, Yanzhang Wei, Jinhua Li, Yasmina Laouar, and Marc D. Basson. "Increased extracellular pressure enhances cancer cell integrin-binding affinity through phosphorylation of β1-integrin at threonine 788/789." American Journal of Physiology-Cell Physiology 296, no. 1 (January 2009): C193—C204. http://dx.doi.org/10.1152/ajpcell.00355.2008.
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Increased extracellular pressure stimulates β1-integrin-dependent cancer cell adhesion. We asked whether pressure-induced adhesion is mediated by changes in β1-integrin binding affinity or avidity and whether these changes are phosphorylation dependent. We evaluated integrin affinity and clustering in human SW620 colon cancer cells by measuring differences in binding between soluble Arg-Gly-Asp (RGD)-Fc ligands and RGD-Fc-F(ab′)2 multimeric complexes under ambient and 15-mmHg increased pressures. Phosphorylation of β1-integrin S785 and T788/9 residues in SW620 and primary malignant colonocytes was assessed in parallel. We further used GD25-β1-integrin-null murine fibroblasts stably transfected with either wild-type β1A-integrin, S785A, TT788/9AA, or T788D mutants to investigate the role of β1-integrin site-specific phosphorylation. SW620 binding of RGD-Fc-F(ab′)2 multimeric complexes, but not soluble RGD-Fc ligands, was sensitive to integrin clustering. RGD-Fc ligand binding was significantly increased under elevated pressure, suggesting that pressure modulates β1-integrin affinity. Pressure stimulated both β1-integrin S785 and T788/9 phosphorylation. GD25-β1A-integrin wild-type and S785A cells displayed an increase in adhesion to fibronectin under elevated pressure, an effect absent in β1-integrin-null and TT788/9AA cells. T788D substitution significantly elevated basal cell adhesion but displayed no further increase under pressure. These results suggest pressure-induced cell adhesion is mediated by β1-integrin T788/9 phosphorylation-dependent changes in integrin binding affinity.
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Legler, D. F., G. Wiedle, F. P. Ross, and B. A. Imhof. "Superactivation of integrin (α)v(β)3 by low antagonist concentrations." Journal of Cell Science 114, no. 8 (April 15, 2001): 1545–53. http://dx.doi.org/10.1242/jcs.114.8.1545.
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Integrins are implicated in cell adhesion, migration and homeostasis. An important feature is their ability to adopt different affinity states that can be regulated by a variety of intra- and extracellular factors. To study affinity modulation of the integrin ectodomain by extracellular factors, we produced a soluble recombinant form of mouse integrin (α)v(β)3 in a mammalian expression system and isolated it to purity. We show that the two transmembrane truncated integrin subunits stably associate to form a functional receptor, soluble recombinant (α)v(β)3. The affinity of this receptor for its ligands vitronectin, fibronectin and fibrinogen can be modulated by the divalent cations magnesium, calcium and manganese. Most importantly, we found that a cyclic RGD-peptide has a biphasic effect on rs(α)v(β)3and native purified (α)v(β)3, with an antagonistic phase at high concentrations, and an agonistic phase at low concentrations. This integrin superactivation by low antagonist concentrations is shown in binding of sr(α)v(β)3 to immobilized ligands by ELISA, and in adhesion of cells that express the chimaeric integrin ligand KISS31 to immobilized rs(α)v(β)3 and native purified (α)v(β)3. Our results indicate that low concentrations of the ligand mimetic cyclo-RGD can result in superactivation of the extracellular domain of integrin (α)v(β)3 to a comparable level as activation by manganese.
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Fagerholm, Susanna C., Tiina J. Hilden, Susanna M. Nurmi, and Carl G. Gahmberg. "Specific integrin α and β chain phosphorylations regulate LFA-1 activation through affinity-dependent and -independent mechanisms." Journal of Cell Biology 171, no. 4 (November 21, 2005): 705–15. http://dx.doi.org/10.1083/jcb.200504016.
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Integrins are adhesion receptors that are crucial to the functions of multicellular organisms. Integrin-mediated adhesion is a complex process that involves both affinity regulation and cytoskeletal coupling, but the molecular mechanisms behind this process have remained incompletely understood. In this study, we report that the phosphorylation of each cytoplasmic domain of the leukocyte function-associated antigen-1 integrin mediates different modes of integrin activation. α Chain phosphorylation on Ser1140 is needed for conformational changes in the integrin after chemokine- or integrin ligand–induced activation or after activation induced by active Rap1 (Rap1V12). In contrast, the β chain Thr758 phosphorylation mediates selective binding to 14-3-3 proteins in response to inside-out activation through the T cell receptor, resulting in cytoskeletal rearrangements. Thus, site-specific phosphorylation of the integrin cytoplasmic domains is important for the dynamic regulation of these complex receptors in cells.
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PICCARDONI, Paola, Stefano MANARINI, Lorenzo FEDERICO, Zsuzsa BAGOLY, Romina PECCE, Nicola MARTELLI, Antonio PICCOLI, Licia TOTANI, Chiara CERLETTI, and Virgilio EVANGELISTA. "SRC-dependent outside-in signalling is a key step in the process of autoregulation of beta2 integrins in polymorphonuclear cells." Biochemical Journal 380, no. 1 (May 15, 2004): 57–65. http://dx.doi.org/10.1042/bj20040151.
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In human PMN (polymorphonuclear cells), challenged by P-selectin, the β2-integrin Mac-1 (macrophage antigen-1) promoted the activation of the SRC (cellular homologue of Rous sarcoma virus oncogenic protein) family members HCK (haematopoietic cell kinase) and LYN (an SRC family protein tyrosine kinase) and phosphorylation of a P-110 (110 kDa protein). SRC kinase activity in turn was necessary for macrophage antigen-1-mediated adhesion [Piccardoni, Sideri, Manarini, Piccoli, Martelli, de Gaetano, Cerletti and Evangelista (2001) Blood 98, 108–116]. This suggested that an SRC-dependent outside-in signalling strengthens the β2-integrin interaction with the ligand. To support this hypothesis further, in the present study, we used the monoclonal antibody KIM127 or manganese to lock β2 integrins in a high-affinity state, and homotypic PMN adhesion was analysed to monitor β2-integrin adhesive function. KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation. Both these processes were abolished by blocking antibodies against the common β2 chain, by a combination of antibodies against αL and αM or by inhibitors of SRC activity. Confocal microscopy showed that activation epitopes were expressed by β2 integrins co-localized with patches of F-actin at the adhesion sites. Blockade of SRC kinases or of actin polymerization prevented clustering of activated integrins as well as F-actin accumulation. FACS analysis showed that SRC inhibitors modified neither basal nor manganese-induced KIM127 binding. An SRC-dependent outside-in signalling initiated by β2 integrins was also required for adhesion triggered by interleukin-8. These results confirm the hypothesis that an SRC-dependent outside-in signalling triggered by high affinity and ligand binding is necessary to stabilize β2-integrin-mediated adhesion. Allowing clustering of activated integrins, SRC might link the high-affinity with the high-avidity state. Proline-rich tyrosine kinase-2 appears to be involved in this process.
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Nieswandt, Bernhard, Markus Moser, Irina Pleines, David Varga-Szabo, Sue Monkley, David Critchley, and Reinhard Fässler. "Loss of talin1 in platelets abrogates integrin activation, platelet aggregation, and thrombus formation in vitro and in vivo." Journal of Experimental Medicine 204, no. 13 (December 17, 2007): 3113–18. http://dx.doi.org/10.1084/jem.20071827.
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Platelet adhesion and aggregation at sites of vascular injury are essential for normal hemostasis but may also lead to pathological thrombus formation, causing diseases such as myocardial infarction or stroke. Heterodimeric receptors of the integrin family play a central role in the adhesion and aggregation of platelets. In resting platelets, integrins exhibit a low affinity state for their ligands, and they shift to a high affinity state at sites of vascular injury. It has been proposed that direct binding of the cytoskeletal protein talin1 to the cytoplasmic domain of the integrin β subunits is necessary and sufficient to trigger the activation of integrins to this high affinity state, but direct in vivo evidence in support of this hypothesis is still lacking. Here, we show that platelets from mice lacking talin1 are unable to activate integrins in response to all known major platelet agonists while other cellular functions are still preserved. As a consequence, mice with talin-deficient platelets display a severe hemostatic defect and are completely resistant to arterial thrombosis. Collectively, these experiments demonstrate that talin is required for inside-out activation of platelet integrins in hemostasis and thrombosis.
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Hadari, Y. R., R. Arbel-Goren, Y. Levy, A. Amsterdam, R. Alon, R. Zakut, and Y. Zick. "Galectin-8 binding to integrins inhibits cell adhesion and induces apoptosis." Journal of Cell Science 113, no. 13 (July 1, 2000): 2385–97. http://dx.doi.org/10.1242/jcs.113.13.2385.
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The interaction of cells with the extracellular matrix regulates cell adhesion, motility, growth, survival and differentiation through integrin-mediated signal transduction. Here we demonstrate that galectin-8, a secreted mammalian (beta)-galactoside binding protein, inhibits adhesion of human carcinoma (1299) cells to plates coated with integrin ligands, and induces cell apoptosis. Pretreatment of the cells with Mn(2+), which increases the affinity of integrins for their ligands, abolished the inhibitory effects of galectin-8. The inhibitory effects of galectin-8 were specific and were not mimicked by plant lectins or other galectins (galectin-1 and galectin-3). In accordance with its anti-adhesive effects, transfection of galectin-8 cDNA into 1299 cells significantly reduced (by 75%) colony formation, when compared to the number of colonies formed by cells transfected with an empty vector. Affinity chromatography over immobilized galectin-8 indicated that few membrane proteins interacted with galectin-8 in a sugar-dependent manner. Microsequencing and western immunoblotting revealed that (alpha)(3)(beta)(1)integrin derived from 1299 as well as other cells (e.g. HeLa and human endothelial cells) is a major galectin-8 binding-protein. Furthermore, immunoprecipitation and immunohistochemical studies suggested that endogenous galectin-8, secreted from 1299 cells, forms complexes with (alpha)(3)(beta)(1) integrins expressed on the surface of 1299 cells. Galectin-8 also interacts with other members of the integrin family, like (alpha)(6)(beta)(1)integrins. In contrast, galectin-8 only minimally interacts with (alpha)(4)or (beta)(3)integrins. We propose that galectin-8 is an integrin binding-protein that interacts to a different extent with several, but not all members of the integrin family. Binding of galectin-8 modulates integrin interactions with the extracellular matrix and thus regulates cell adhesion and cell survival.
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Vasconcelos, Disraeli, Beatriz Chaves, Aline Albuquerque, Luca Andrade, Andrielly Henriques, Geraldo Sartori, Wilson Savino, Ernesto Caffarena, and João Herminio Martins-Da-Silva. "Development of New Potential Inhibitors of β1 Integrins through In Silico Methods—Screening and Computational Validation." Life 12, no. 7 (June 22, 2022): 932. http://dx.doi.org/10.3390/life12070932.
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Integrins are transmembrane receptors that play a critical role in many biological processes which can be therapeutically modulated using integrin blockers, such as peptidomimetic ligands. This work aimed to develop new potential β1 integrin antagonists using modeled receptors based on the aligned crystallographic structures and docked with three lead compounds (BIO1211, BIO5192, and TCS2314), widely known as α4β1 antagonists. Lead-compound complex optimization was performed by keeping intact the carboxylate moiety of the ligand, adding substituents in two other regions of the molecule to increase the affinity with the target. Additionally, pharmacokinetic predictions were performed for the ten best ligands generated, with the lowest docking interaction energy obtained for α4β1 and BIO5192. Results revealed an essential salt bridge between the BIO5192 carboxylate group and the Mg2+ MIDAS ion of the integrin. We then generated more than 200 new BIO5192 derivatives, some with a greater predicted affinity to α4β1. Furthermore, the significance of retaining the pyrrolidine core of the ligand and increasing the therapeutic potential of the new compounds is emphasized. Finally, one novel molecule (1592) was identified as a potential drug candidate, with appropriate pharmacokinetic profiles, similar dynamic behavior at the integrin interaction site compared with BIO5192, and a higher predicted affinity to VLA-4.
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Laudanna, Carlo, and Ronen Alon. "Right on the spot." Thrombosis and Haemostasis 95, no. 01 (2006): 5–11. http://dx.doi.org/10.1160/th05-07-0482.
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SummaryThe arrest of rolling leukocytes on various target vascular beds is crucial for their recruitment at inflammatory sites and secondary lymphoid tissues. Leukocyte arrest is predominantly mediated by integrins interacting with either constitutive or inducible endothelial ligands. Integrins are cytoskeletally regulated heterodimers maintained in largely low affinity conformational states on circulating leukocytes. For arrest to occur, the affinity of integrin heterodimers must be enhanced in situ upon leukocyte encounter with proper endothelial-displayed chemokines or chemoattractants which bind and signal through specific G-protein coupled receptors (GPCRs) on the leukocyte surface. Recent studies suggest that this integrin activation involves rapid conformational alterations locally induced at confined leukocyte-endothelial contact sites. Following binding, integrin microclustering reinforced by associations with the cortical actin cytoskeleton further enhances integrin-mediated adhesiveness under shear stress. These events are controlled by complex signaling events, involving a series of small GTPases, as well as protein and lipid kinases which are triggered by chemokine bound GPCRs. To rapidly mediate this specialized function, subsets of signaling proteins and their specific targets are thought to preexist in pre-assembled multi-molecular complexes or signalosomes. Recent in vitro dissection of chemokine-triggered integrin activation on lymphocytes and neutrophils suggests that these signalosomes may vary both in composition and mode of activity between different immune cell types and distinct integrins. We review in this article recent findings on key elements implicated in chemokine triggering of integrin activation on rolling leukocytes, and discuss the possible existence of preformed proadhesive signaling networks in different subsets of leukocytes.
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Asokan, Aravind, Julie B. Hamra, Lakshmanan Govindasamy, Mavis Agbandje-McKenna, and Richard J. Samulski. "Adeno-Associated Virus Type 2 Contains an Integrin α5β1 Binding Domain Essential for Viral Cell Entry." Journal of Virology 80, no. 18 (September 15, 2006): 8961–69. http://dx.doi.org/10.1128/jvi.00843-06.
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ABSTRACT Integrins have been implicated as coreceptors in the infectious pathways of several nonenveloped viruses. For example, adenoviruses are known to interact with αV integrins by virtue of a high-affinity arginine-glycine-aspartate (RGD) domain present in the penton bases of the capsids. In the case of adeno-associated virus type 2 (AAV2), which lacks this RGD motif, integrin αVβ5 has been identified as a coreceptor for cellular entry. However, the molecular determinants of AAV2 capsid-integrin interactions and the potential exploitation of alternative integrins as coreceptors by AAV2 have not been established thus far. In this report, we demonstrate that integrin α5β1 serves as an alternative coreceptor for AAV2 infection in human embryonic kidney 293 cells. Such interactions appear to be mediated by a highly conserved domain that contains an asparagine-glycine-arginine (NGR) motif known to bind α5β1 integrin with moderate affinity. The mutation of this domain reduces transduction efficiency by an order of magnitude relative to that of wild-type AAV2 vectors in vitro and in vivo. Further characterization of mutant and wild-type AAV2 capsids through transduction assays in cell lines lacking specific integrins, cell adhesion studies, and cell surface/solid-phase binding assays confirmed the role of the NGR domain in promoting AAV2-integrin interactions. Molecular modeling studies suggest that NGR residues form a surface loop close to the threefold axis of symmetry adjacent to residues previously implicated in binding heparan sulfate, the primary receptor for AAV2. The aforementioned results suggest that the internalization of AAV2 in 293 cells might follow a “click-to-fit” mechanism that involves the cooperative binding of heparan sulfate and α5β1 integrin by the AAV2 capsids.
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Ye, Feng, Guiqing Hu, Dianne Taylor, Boris Ratnikov, Andrey A. Bobkov, Mark A. McLean, Stephen G. Sligar, Kenneth A. Taylor, and Mark H. Ginsberg. "Recreation of the terminal events in physiological integrin activation." Journal of Cell Biology 188, no. 1 (January 4, 2010): 157–73. http://dx.doi.org/10.1083/jcb.200908045.
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Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin αIIbβ3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin αIIbβ3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.
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Otey, C. A., F. M. Pavalko, and K. Burridge. "An interaction between alpha-actinin and the beta 1 integrin subunit in vitro." Journal of Cell Biology 111, no. 2 (August 1, 1990): 721–29. http://dx.doi.org/10.1083/jcb.111.2.721.
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A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely alpha-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect the additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of beta 1 integrin and affinity chromatography to identify additional integrin-binding proteins. Here we report our finding of an interaction between the cytoplasmic domain of beta 1 integrin and the actin-binding protein alpha-actinin. Beta 1-integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at approximately 100 kD was identified by immunoblot analysis as alpha-actinin. Solid phase binding assays indicated that alpha-actinin bound specifically and directly to the beta 1 peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a beta 1 integrin) or the platelet integrin glycoprotein IIb/IIIa complex (a beta 3 integrin), binding of alpha-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the beta 1 peptide. alpha-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-alpha-actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations.
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Isberg, R. R., and P. Barnes. "Subversion of integrins by enteropathogenic Yersinia." Journal of Cell Science 114, no. 1 (January 1, 2001): 21–28. http://dx.doi.org/10.1242/jcs.114.1.21.
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Enteropathogenic Yersinia are gram-negative bacterial species that translocate from the lumen of the intestine and are able to grow within deep tissue sites. During the earliest stages of disease, the organism is able to bind integrin receptors that are presented on the apical surface of M cells in the intestine, which allows its internalization and subsequent translocation into regional lymph nodes. The primary integrin substrate is the outer-membrane protein invasin, which binds with extraordinarily high affinity to at least five different integrins that have the (beta)(1) chain. Bacterial uptake into host cells is modulated by the affinity of receptor-substrate interaction, receptor concentration and the ability of the substrate to aggregate target receptors.
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De Mets, Richard, Irene Wang, Martial Balland, Christiane Oddou, Philippe Moreau, Bertrand Fourcade, Corinne Albiges-Rizo, Antoine Delon, and Olivier Destaing. "Cellular tension encodes local Src-dependent differential β1 and β3 integrin mobility." Molecular Biology of the Cell 30, no. 2 (January 15, 2019): 181–90. http://dx.doi.org/10.1091/mbc.e18-04-0253.
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Integrins are transmembrane receptors that have a pivotal role in mechanotransduction processes by connecting the extracellular matrix to the cytoskeleton. Although it is well established that integrin activation/inhibition cycles are due to highly dynamic interactions, whether integrin mobility depends on local tension and cytoskeletal organization remains surprisingly unclear. Using an original approach combining micropatterning on glass substrates to induce standardized local mechanical constraints within a single cell with temporal image correlation spectroscopy, we measured the mechanosensitive response of integrin mobility at the whole cell level and in adhesion sites under different mechanical constraints. Contrary to β1 integrins, high tension increases β3 integrin residence time in adhesive regions. Chimeric integrins and structure–function studies revealed that the ability of β3 integrins to specifically sense local tensional organization is mostly encoded by its cytoplasmic domain and is regulated by tuning the affinity of its NPXY domains through phosphorylation by Src family kinases.
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Peter, K., and T. E. O'Toole. "Modulation of cell adhesion by changes in alpha L beta 2 (LFA-1, CD11a/CD18) cytoplasmic domain/cytoskeleton interaction." Journal of Experimental Medicine 181, no. 1 (January 1, 1995): 315–26. http://dx.doi.org/10.1084/jem.181.1.315.
Full textAbstract:
The integrin alpha L beta 2 (leukocyte function-associated molecule 1, CD11a/CD18) mediates activation-dependent adhesion of leukocytes. The cytoplasmic domains of alpha L beta 2 have been demonstrated to modulate adhesiveness of alpha L beta 2. Affinity changes of alpha L beta 2 for its ligand or postreceptor events can be responsible for this modulation of adhesiveness. To investigate the possible role of the alpha L beta 2 cytoplasmic domains in postreceptor events we constructed cDNA encoding chimeric proteins with intracellular alpha L beta 2 domains, which are responsible for alpha L beta 2 specific intracellular interactions, and extracellular alpha IIIb beta 3 (GP IIb/IIIa) domains, which allow the assessment of the receptor affinity state. The cDNA was stably transfected in Chinese hamster ovary cells and chimeric heterodimer formation proven by immunoprecipitations and flow cytometry. The chimeric receptors mediate adhesion to immobilized fibrinogen, and this adhesion is increased by phorbol myristate acetate and abolished by cytochalasin D. However, neither treatment affects the affinity state of the chimeric receptor, suggesting involvement of the cytoskeleton in the regulation of alpha L beta 2 mediated cell adhesion. To exclude the possibility of postoccupancy affinity changes of the chimeric receptors, we locked the receptors into a high affinity state by creating a deletion variant. The region deleted (VGFFK) is highly conserved in integrin alpha subunit cytoplasmic domains. Cotransfection of this deletion variant with a beta subunit truncation (beta 3 delta 724) and a triple mutation at 758-760 (TTT to AAA) of beta 2 abolishes adhesion without changing the affinity state. A single mutation (TTT to TAT) reduces adhesion by half without affinity change. Scanning electron microscopy reveals impaired spreading of these truncated/mutated chimeras. Immunofluorescence microscopy demonstrates a correlation between impaired adhesion and a decrease in the ability to form focal adhesions and to organize the cytoskeleton into stress fibers. These results describe the integrin/cytoskeleton interaction, the organization of the cytoskeleton, and cell spreading as postreceptor events modulating alpha L beta 2 cytoplasmic domain mediated cell adhesion. Furthermore, we demonstrate that the cytoplasmic domain of the beta 2 subunit, and within it the TTT region, are required for these postreceptor events. Additionally, we present a new approach, using deletion variants to lock integrins in a high affinity state without interfering with the investigated integrin/cytoskeleton interaction. This approach may be generally useful to investigate the role of postreceptor events in integrin-mediated cell adhesion and migration.
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Pasvolsky, Ronit, Sara W. Feigelson, Sara Sebnem Kilic, Amos J. Simon, Guy Tal-Lapidot, Valentin Grabovsky, Jill R. Crittenden, et al. "A LAD-III syndrome is associated with defective expression of the Rap-1 activator CalDAG-GEFI in lymphocytes, neutrophils, and platelets." Journal of Experimental Medicine 204, no. 7 (June 18, 2007): 1571–82. http://dx.doi.org/10.1084/jem.20070058.
Full textAbstract:
Leukocyte and platelet integrins rapidly alter their affinity and adhesiveness in response to various activation (inside-out) signals. A rare leukocyte adhesion deficiency (LAD), LAD-III, is associated with severe defects in leukocyte and platelet integrin activation. We report two new LAD cases in which lymphocytes, neutrophils, and platelets share severe defects in β1, β2, and β3 integrin activation. Patients were both homozygous for a splice junction mutation in their CalDAG-GEFI gene, which is a key Rap-1/2 guanine exchange factor (GEF). Both mRNA and protein levels of the GEF were diminished in LAD lymphocytes, neutrophils, and platelets. Consequently, LAD-III platelets failed to aggregate because of an impaired αIIbβ3 activation by key agonists. β2 integrins on LAD-III neutrophils were unable to mediate leukocyte arrest on TNFα-stimulated endothelium, despite normal selectin-mediated rolling. In situ subsecond activation of neutrophil β2 integrin adhesiveness by surface-bound chemoattractants and of primary T lymphocyte LFA-1 by the CXCL12 chemokine was abolished. Chemokine inside-out signals also failed to stimulate lymphocyte LFA-1 extension and high affinity epitopes. Chemokine-triggered VLA-4 adhesiveness in T lymphocytes was partially defective as well. These studies identify CalDAG-GEFI as a critical regulator of inside-out integrin activation in human T lymphocytes, neutrophils, and platelets.
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Dogic, D., P. Rousselle, and M. Aumailley. "Cell adhesion to laminin 1 or 5 induces isoform-specific clustering of integrins and other focal adhesion components." Journal of Cell Science 111, no. 6 (March 15, 1998): 793–802. http://dx.doi.org/10.1242/jcs.111.6.793.
Full textAbstract:
Laminin 1 (alpha1beta1gamma1) and laminin 5 (alpha3beta3gamma2) induce cell adhesion with different involvement of integrins: both are ligands for the alpha6beta1 integrin, while alpha3beta1 integrin has affinity for laminin 5 only. These two laminin isoforms therefore provide good models to investigate whether alpha3beta1 and alpha6beta1 integrins play different roles in signal transduction and in focal adhesion formation. Laminin 1 or 5 induced adhesion of normal human skin fibroblasts to a similar extent but promoted different overall cell shapes. On laminin 1 the fibroblasts formed mainly filopodia-like structures, while on laminin 5 they developed lamellipodias. Staining of fibrillar actin with fluorescein-phalloidin revealed a similar organisation of the actin cytoskeleton on both substrates. However, integrin subunits and several cytoskeletal linker proteins, including vinculin, talin, and paxillin, showed an isoform-specific arrangement into focal adhesions. On laminin 1 they were recruited into thick and short aggregates localized at the termini of actin stress fibers, while on laminin 5 they appeared as dots or streaks clustered on a long portion of actin microfilaments. To test whether the differing affinity of laminin 1 or 5 for alpha3beta1 integrin would explain the formation of morphologically different focal adhesions, cells were seeded on laminin 1 under conditions in which alpha3beta1 integrins were occupied by a function-blocking antibody. This resulted in the formation of focal adhesions similar to that observed on laminin 5, where the integrin is occupied by its natural ligand. These results provide the first evidence for a cross-talk between alpha3beta1 and alpha6beta1 integrins and indicate that occupancy of alpha3beta1 integrins results in a trans-dominant regulation of alpha6beta1 integrin clustering and of focal adhesions. It suggests that recruitment of integrins and cytoskeletal linker proteins are laminin isoform-specific and that tissue specific expression of laminin isoforms might modulate cell behavior by the activation of distinct sets of integrins and by the induction of distinct molecular assemblies within the cell adhesion signaling complexes.
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Haling, Jacob R., Susan J. Monkley, David R. Critchley, and Brian G. Petrich. "Talin-dependent integrin activation is required for fibrin clot retraction by platelets." Blood 117, no. 5 (February 3, 2011): 1719–22. http://dx.doi.org/10.1182/blood-2010-09-305433.
Full textAbstract:
Abstract Talin functions both as a regulator of integrin affinity and as an important mechanical link between integrins and the cytoskeleton. Using genetic deletion of talin, we show for the first time that the capacity of talin to activate integrins is required for fibrin clot retraction by platelets. To further dissect which talin functions are required for this process, we tested clot retraction in platelets expressing a talin1(L325R) mutant that binds to integrins, but exhibits impaired integrin activation ascribable to disruption of the interaction between talin and the membrane-proximal region (MPR) in the β-integrin cytoplasmic domain. Talin-deficient and talin1(L325R) platelets were defective in retracting fibrin clots. However, the defect in clot retraction in talin1(L325R) platelets, but not talin-deficient platelets, was rescued by extrinsically activating integrins with manganese, thereby proving that integrin activation is required and showing that talin1(L325R) can form functional links to the actin cytoskeleton.