The Experts below are selected from a list of 25935 Experts worldwide ranked by ideXlab platform
Mark H Ginsberg - One of the best experts on this subject based on the ideXlab platform.
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optogenetic based localization of talin to the plasma membrane promotes Activation of β3 Integrins
2021Co-Authors: Zhongji Liao, Mark H Ginsberg, Alexandre R Gingras, Frederic Lagarrigue, Sanford J ShattilAbstract:Interaction of talin with the cytoplasmic tails of Integrin β triggers Integrin Activation, leading to an increase of Integrin affinity/avidity for extracellular ligands. In talin knockout mice, loss of talin interaction with platelet Integrin αIIbβ3 causes a severe hemostatic defect, and loss of talin interaction with endothelial cell Integrin αVβ3 affects angiogenesis. In normal cells, talin is auto-inhibited and localized in the cytoplasm. Here we employed an optogenetic platform to assess whether recruitment of full-length talin to the plasma membrane was sufficient to induce Integrin Activation. A dimerization module (CRY2 fused to the N-terminus of talin; CIBN-CAAX) responsive to 450 nm (blue) light was inserted into CHO cells and endothelial cells also expressing αIIbβ3 or αVβ3, respectively. Thus, exposure of the cells to blue light caused a rapid and reversible recruitment of CRY2-talin to the CIBN-CAAX-decorated plasma membrane. This resulted in β3 Integrin Activation in both cell types, as well as increasing migration of the endothelial cells. However, membrane recruitment of talin was not sufficient for Integrin Activation, as membrane-associated Rap1-GTP was also required. Moreover, talin mutations that interfered with its direct binding to Rap1 abrogated β3 Integrin Activation. Altogether, these results define a role for the plasma membrane recruitment of talin in β3 Integrin Activation, and they suggest a nuanced sequence of events thereafter involving Rap1-GTP.
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distinct Integrin Activation pathways for effector and regulatory t cell trafficking and function
2021Co-Authors: Hao Sun, Frederic Lagarrigue, Miguel Alejandro Lopezramirez, Hsin Wang, Zhichao Fan, John T Chang, Mark H GinsbergAbstract:Integrin Activation mediates lymphocyte trafficking and immune functions. Conventional T cell (Tconv cell) Integrin Activation requires Rap1-interacting adaptor molecule (RIAM). Here, we report that Apbb1ip-/- (RIAM-null) mice are protected from spontaneous colitis due to IL-10 deficiency, a model of inflammatory bowel disease (IBD). Protection is ascribable to reduced accumulation and homing of Tconv cells in gut-associated lymphoid tissue (GALT). Surprisingly, there are abundant RIAM-null regulatory T cells (T reg cells) in the GALT. RIAM-null T reg cells exhibit normal homing to GALT and lymph nodes due to preserved Activation of Integrins αLβ2, α4β1, and α4β7. Similar to Tconv cells, T reg cell Integrin Activation and immune function require Rap1; however, lamellipodin (Raph1), a RIAM paralogue, compensates for RIAM deficiency. Thus, in contrast to Tconv cells, RIAM is dispensable for T reg cell Integrin Activation and suppressive function. In consequence, inhibition of RIAM can inhibit spontaneous Tconv cell-mediated autoimmune colitis while preserving T reg cell trafficking and function.
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a rap1 binding site and lipid dependent helix in talin f1 domain cooperate in Integrin Activation
2018Co-Authors: Alexandre R Gingras, Frederic Lagarrigue, Monica N Cuevas, Marcus Zorovich, Andrew J Valadez, Miguel Alejandro Lopezramirez, Nicolas Seban, Wilma Mclaughlin, Mark H GinsbergAbstract:Rap1 GTPases bind effectors, such as RIAM, to enable talin1 to induce Integrin Activation. In addition, Rap1 binds directly to the talin1 F0 domain (F0); however, this interaction makes a negligible contribution to Integrin Activation in CHO cells or platelets. Here, we show that talin1 F1 domain contains a previously undetected Rap1 binding site of similar affinity to that in F0. A structure-guided point mutant (R118E) in F1, which blocks Rap1 binding, abolishes the capacity of Rap1 to potentiate talin1-induced Integrin Activation. The capacity of F1 to mediate Rap1-dependent Integrin Activation depends on a unique loop in F1 that transforms into an amphipathic helix upon binding to membrane lipids. Basic membrane-facing residues of this helix are critical as charge reversal mutations led to dramatic suppression of talin1-dependent Activation. Thus, a novel Rap1 binding site and a lipid-dependent amphipathic helix in talin1 F1 cooperate to enable a direct Rap1-talin1 interaction to cause Integrin Activation.
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the rap1 riam talin axis of Integrin Activation and blood cell function
2016Co-Authors: Frederic Lagarrigue, Chungho Kim, Mark H GinsbergAbstract:Integrin adhesion receptors mediate the adhesion of blood cells, such as leukocytes, to other cells, such as endothelial cells. Integrins also are critical for anchorage of hematopoietic precursors to the extracellular matrix. Blood cells can dynamically regulate the affinities of Integrins for their ligands ("Activation"), an event central to their functions. Here we review recent progress in understanding the mechanisms of Integrin Activation with a focus on the functions of blood cells. We discuss how talin binding to the Integrin β cytoplasmic domain, in conjunction with the plasma membrane, induces long-range allosteric rearrangements that lead to Integrin Activation. Second, we review our understanding of how signaling events, particularly those involving Rap1 small guanosine triphosphate (GTP)hydrolases, can regulate the talin-Integrin interaction and resulting Activation. Third, we review recent findings that highlight the role of the Rap1-GTP-interacting adapter molecule (RIAM), encoded by the APBB1IP gene, in leukocyte Integrin Activation and consequently in leukocyte trafficking.
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blocking neutrophil Integrin Activation prevents ischemia reperfusion injury
2015Co-Authors: Tadayuki Yago, Mark H Ginsberg, Brian G Petrich, Nan Zhang, Bojing Shao, Rodger P MceverAbstract:Neutrophil recruitment, mediated by β2 Integrins, combats pyogenic infections but also plays a key role in ischemia–reperfusion injury and other inflammatory disorders. Talin induces allosteric rearrangements in Integrins that increase affinity for ligands (Activation). Talin also links Integrins to actin and other proteins that enable formation of adhesions. Structural studies have identified a talin1 mutant (L325R) that perturbs Activation without impairing talin’s capacity to link Integrins to actin and other proteins. Here, we found that mice engineered to express only talin1(L325R) in myeloid cells were protected from renal ischemia–reperfusion injury. Dissection of neutrophil function in vitro and in vivo revealed that talin1(L325R) neutrophils had markedly impaired chemokine-induced, β2 Integrin–mediated arrest, spreading, and migration. Surprisingly, talin1(L325R) neutrophils exhibited normal selectin-induced, β2 Integrin–mediated slow rolling, in sharp contrast to the defective slow rolling of neutrophils lacking talin1 or expressing a talin1 mutant (W359A) that blocks talin interaction with Integrins. These studies reveal the importance of talin-mediated Activation of Integrins for renal ischemia–reperfusion injury. They further show that neutrophil arrest requires talin recruitment to and Activation of Integrins. However, although neutrophil slow rolling requires talin recruitment to Integrins, talin-mediated Integrin Activation is dispensable.
Alexander Zarbock - One of the best experts on this subject based on the ideXlab platform.
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Integrin Activation by p rex1 is required for selectin mediated slow leukocyte rolling and intravascular crawling
2013Co-Authors: Jan M Herter, Jan Rossaint, Helena Block, Heidi C E Welch, Alexander ZarbockAbstract:Integrin Activation is essential for the function of leukocytes. Impaired Integrin Activation on leukocytes is the hallmark of the leukocyte adhesion deficiency syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. In inflammation, leukocytes collect different signals during the contact with the microvasculature, which activate signaling pathways leading to Integrin Activation and leukocyte recruitment. We report the role of P-Rex1, a Rac-specific guanine nucleotide exchanging factor, in Integrin Activation and leukocyte recruitment. We find that P-Rex1 is required for inducing selectin-mediated lymphocyte function-associated antigen-1 (LFA-1) extension that corresponds to intermediate affinity and induces slow leukocyte rolling, whereas P-Rex1 is not involved in the induction of the high-affinity conformation of LFA-1 obligatory for leukocyte arrest. Furthermore, we demonstrate that P-Rex1 is involved in Mac-1-dependent intravascular crawling. In vivo, both LFA-1-dependent slow rolling and Mac-1-dependent crawling are defective in P-Rex1(-/-) leukocytes, whereas chemokine-induced arrest and postadhesion strengthening remain intact in P-Rex1-deficient leukocytes. Rac1 is involved in E-selectin-mediated slow rolling and crawling. In vivo, in an ischemia-reperfusion-induced model of acute kidney injury, abolished selectin-mediated Integrin Activation contributed to decreased neutrophil recruitment and reduced kidney damage in P-Rex1-deficient mice. We conclude that P-Rex1 serves distinct functions in LFA-1 and Mac-1 Activation.
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Integrin Activation by p rex1 is required for selectin mediated slow leukocyte rolling and intravascular crawling
2013Co-Authors: Jan M Herter, Jan Rossaint, Helena Block, Heidi C E Welch, Alexander ZarbockAbstract:Integrin Activation is essential for the function of leukocytes. Impaired Integrin Activation on leukocytes is the hallmark of the leukocyte adhesion deficiency syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. In inflammation, leukocytes collect different
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Leukocyte Integrin Activation and deActivation: novel mechanisms of balancing inflammation
2012Co-Authors: Alexander Zarbock, Tibor Kempf, Kai C. Wollert, Dietmar VestweberAbstract:Leukocyte recruitment into tissue forms the basis of immune surveillance and direct immune defense. It proceeds in a cascade-like fashion. The first contact of leukocytes with the endothelium is mediated by selectins and their counter receptors, followed by rolling and Integrin-mediated arrest. While rolling, neutrophils collect different inflammatory signals which can activate several signaling pathways leading to leukocyte adhesion to the endothelium and transmigration through the blood vessel wall into the inflamed tissue. Whereas inflammatory reactions are beneficial and necessary for host defense, they need to be balanced and controlled to prevent harmful consequences and tissue destruction. In this article, we discuss the different signaling pathways that ensure rapid and efficient Integrin Activation on leukocytes. In addition, we report on a recently identified novel endogenous mechanism that counteracts and balances Integrin Activation, thereby limiting leukocyte recruitment and the extent of inflammation. Further investigation of this new mechanism may allow providing new approaches for the development of the next generation of anti-inflammatory drugs.
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gdf 15 is an inhibitor of leukocyte Integrin Activation required for survival after myocardial infarction in mice
2011Co-Authors: Tibor Kempf, Anika Stadtmann, Jan Rossaint, Alexander Zarbock, Stefan Butz, Christian Widera, Matteo Bolominivittori, Mortimer Korfklingebiel, Christian L Napp, Birte HansenAbstract:Inflammatory cell recruitment after myocardial infarction needs to be tightly controlled to permit infarct healing while avoiding fatal complications such as cardiac rupture. Growth differentiation factor-15 (GDF-15), a transforming growth factor-β (TGF-β)-related cytokine, is induced in the infarcted heart of mice and humans. We show that coronary artery ligation in Gdf15-deficient mice led to enhanced recruitment of polymorphonuclear leukocytes (PMNs) into the infarcted myocardium and an increased incidence of cardiac rupture. Conversely, infusion of recombinant GDF-15 repressed PMN recruitment after myocardial infarction. In vitro, GDF-15 inhibited PMN adhesion, arrest under flow and transendothelial migration. Mechanistically, GDF-15 counteracted chemokine-triggered conformational Activation and clustering of β(2) Integrins on PMNs by activating the small GTPase Cdc42 and inhibiting Activation of the small GTPase Rap1. Intravital microscopy in vivo in Gdf15-deficient mice showed that Gdf-15 is required to prevent excessive chemokine-activated leukocyte arrest on the endothelium. Genetic ablation of β(2) Integrins in myeloid cells rescued the mortality of Gdf15-deficient mice after myocardial infarction. To our knowledge, GDF-15 is the first cytokine identified as an inhibitor of PMN recruitment by direct interference with chemokine signaling and Integrin Activation. Loss of this anti-inflammatory mechanism leads to fatal cardiac rupture after myocardial infarction.
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tyrosine kinase btk regulates e selectin mediated Integrin Activation and neutrophil recruitment by controlling phospholipase c plc γ2 and pi3kγ pathways
2010Co-Authors: Helena Mueller, Anika Stadtmann, Emilio Hirsch, Demin Wang, Alexander Zarbock, Klaus Ley, Hugo Van AkenAbstract:Selectins mediate leukocyte rolling, trigger β2-Integrin Activation, and promote leukocyte recruitment into inflamed tissue. E-selectin binding to P-selectin glycoprotein ligand 1 (PSGL-1) leads to Activation of an immunoreceptor tyrosine-based Activation motif (ITAM)–dependent pathway, which in turn activates the spleen tyrosine kinase (Syk). However, the signaling pathway linking Syk to Integrin Activation after E-selectin engagement is unknown. To identify the pathway, we used different gene-deficient mice in autoperfused flow chamber, intravital microscopy, peritonitis, and biochemical studies. We report here that the signaling pathway downstream of Syk divides into a phospholipase C (PLC) γ2– and phosphoinositide 3-kinase (PI3K) γ–dependent pathway. The Tec family kinase Bruton tyrosine kinase (Btk) is required for activating both pathways, generating inositol-3,4,5-trisphosphate (IP3), and inducing E-selectin–mediated slow rolling. Inhibition of this signal-transduction pathway diminished Gαi-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster. Gαi-independent neutrophil recruitment into the inflamed peritoneal cavity was reduced in Btk−/− and Plcg2−/− mice. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by E-selectin engagement.
Jun Qin - One of the best experts on this subject based on the ideXlab platform.
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Rap1 and membrane lipids cooperatively recruit talin to trigger Integrin Activation.
2019Co-Authors: Thomas Bromberger, Jun Qin, Liang Zhu, Sarah Klapproth, Markus MoserAbstract:Recruitment and tethering of talin to the plasma membrane initiate the process of Integrin Activation. Multiple factors including the Rap1 proteins, RIAM (also known as APBB1IP) and PIP2 bind talin proteins and have been proposed to regulate these processes, but not systematically analyzed. By expressing specific talin mutants into talin-null fibroblasts, we show that binding of the talin F0 domain to Rap1 synergizes with membrane lipid binding of the talin F2 domain during talin membrane targeting and Integrin Activation, whereas the interaction of the talin rod with RIAM was dispensable. We also characterized a second Rap1-binding site within the talin F1 domain by detailed NMR analysis. Interestingly, while talin F1 exhibited significantly weaker Rap1-binding affinity than talin F0, expression of a talin F1 Rap1-binding mutant inhibited cell adhesion, spreading, talin recruitment and Integrin Activation similarly to the talin F0 Rap1-binding mutant. Moreover, the defects became significantly stronger when both Rap1-binding sites were mutated. In conclusion, our data suggest a model in which cooperative binding of Rap1 to the talin F0 and F1 domains synergizes with membrane PIP2 binding to spatiotemporally position and activate talins to regulate Integrin activity.
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direct rap1 talin1 interaction regulates platelet and neutrophil Integrin activity in mice
2018Co-Authors: Thomas Bromberger, Jun Qin, Liang Zhu, Sarah Klapproth, Markus Sperandio, Ina Rohwedder, L Mittmann, Christoph A Reichel, Markus MoserAbstract:Targeting Talin1 to the plasma membrane is a crucial step in Integrin Activation, which in leukocytes is mediated by a Rap1/RIAM/Talin1 pathway, whereas in platelets, it is RIAM independent. Recent structural, biochemical, and cell biological studies have suggested direct Rap1/Talin1 interaction as an alternative mechanism to recruit Talin1 to the membrane and induce Integrin Activation. To test whether this pathway is of relevance in vivo, we generated Rap1 binding-deficient Talin1 knockin (Tln13mut) mice. Although Tln13mut mice showed no obvious abnormalities, their platelets exhibited reduced Integrin Activation, aggregation, adhesion, and spreading, resulting in prolonged tail-bleeding times and delayed thrombus formation and vessel occlusion in vivo. Surprisingly, neutrophil adhesion to different Integrin ligands and β2 Integrin-dependent phagocytosis were also significantly impaired, which caused profound leukocyte adhesion and extravasation defects in Tln13mut mice. In contrast, macrophages exhibited no defect in adhesion or spreading despite reduced Integrin Activation. Taken together, our findings suggest that direct Rap1/Talin1 interaction is of particular importance in regulating the activity of different Integrin classes expressed on platelets and neutrophils, which both depend on fast and dynamic Integrin-mediated responses.
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structural basis of phosphoinositide binding to kindlin 2 protein pleckstrin homology domain in regulating Integrin Activation
2011Co-Authors: Jianmin Liu, Koichi Fukuda, Edward F Plow, Jamila Hirbawi, Xian Mao, Jun QinAbstract:Kindlins are a subclass of FERM-containing proteins that have recently emerged as key regulators of Integrin receptor Activation and signaling. As compared with the conventional FERM domain, the kindlin FERM domain contains an inserted pleckstrin homology (PH) domain that recognizes membrane phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). Using NMR spectroscopy, we show that PIP3 site-specifically binds to kindlin-2 PH with substantial chemical shift changes that are much larger than PIP2. This suggests an enhanced association of kindlin-2 with membrane as mediated by PIP3 upon its conversion from PIP2 by phosphoinositide-3 kinase, a known regulator of Integrin Activation. We determined the NMR structure of the kindlin-2 PH domain bound to the head group of PIP3, inositol 1,3,4,5-tetraphosphate (IP4). The structure reveals a canonical PH domain fold, yet with a distinct IP4 binding pocket that appears highly conserved for the kindlin family members. Functional experiments demonstrate that although wild type kindlin-2 is capable of cooperating with Integrin activator talin to induce synergistic Integrin αIIbβ3 Activation, this ability is significantly impaired for a phosphoinositide binding-defective kindlin-2 mutant. These results define a specific PIP3 recognition mode for the kindlin PH domain. Moreover, they shed light upon a mechanism as to how the PH domain mediates membrane engagement of kindlin-2 to promote its binding to Integrin and cooperation with talin for regulation of Integrin Activation.
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kindlin 2 regulates podocyte adhesion and fibronectin matrix deposition through interactions with phosphoinositides and Integrins
2011Co-Authors: Xiaohua Shi, Hannu Larjava, M Saleem, Sanford J Shattil, Koichi Fukuda, Jun Qin, Matthias KretzlerAbstract:Kindlin-2 is a FERM and PH domain-containing Integrin-binding protein that is emerging as an important regulator of Integrin Activation. How kindlin-2 functions in Integrin Activation, however, is not known. We report here that kindlin-2 interacts with multiple phosphoinositides, preferentially with phosphatidylinositol 3,4,5-trisphosphate. Although Integrin-binding is essential for focal adhesion localization of kindlin-2, phosphoinositide-binding is not required for this process. Using biologically and clinically relevant glomerular podocytes as a model system, we show that Integrin Activation and dependent processes are tightly regulated by kindlin-2: depletion of kindlin-2 reduced Integrin Activation, matrix adhesion and fibronectin matrix deposition, whereas overexpression of kindlin-2 promoted these processes. Furthermore, we provide evidence showing that kindlin-2 is involved in phosphoinositide-3-kinase-mediated regulation of podocyte-matrix adhesion and fibronectin matrix deposition. Mechanistically, kindlin-2 promotes Integrin Activation and Integrin-dependent processes through interacting with both Integrins and phosphoinositides. TGF-β1, a mediator of progressive glomerular failure, markedly increased the level of kindlin-2 and fibronectin matrix deposition, and the latter process was reversed by depletion of kindlin-2. Our results reveal important functions of kindlin-2 in the regulation of podocyte-matrix adhesion and matrix deposition and shed new light on the mechanism whereby kindlin-2 functions in these processes.
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migfilin a molecular switch in regulation of Integrin Activation
2009Co-Authors: Sujay Subbayya Ithychanda, Edward F Plow, Xiaoxia Wang, Mitali Das, Keyang Ding, Sudhiranjan Gupta, Jun QinAbstract:The linkage of heterodimeric (α/β) Integrin receptors with their extracellular matrix ligands and intracellular actin cytoskeleton is a fundamental step for controlling cell adhesion and migration. Binding of the actin-linking protein, talin, to Integrin β cytoplasmic tails (CTs) induces high affinity ligand binding (Integrin Activation), whereas binding of another actin-linking protein, filamin, to the Integrin β CTs negatively regulates this process by blocking the talin-Integrin interaction. Here we show structurally that migfilin, a novel cytoskeletal adaptor highly enriched in the Integrin adhesion sites, strongly interacts with the same region in filamin where Integrin β CTs bind. We further demonstrate that the migfilin interaction dissociates filamin from Integrin and promotes the talin/Integrin binding and Integrin Activation. Migfilin thus acts as a molecular switch to disconnect filamin from Integrin for regulating Integrin Activation and dynamics of extracellular matrix-actin linkage.
David A Calderwood - One of the best experts on this subject based on the ideXlab platform.
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nanopatterning reveals an ecm area threshold for focal adhesion assembly and force transmission that is regulated by Integrin Activation and cytoskeleton tension
2012Co-Authors: Sean R Coyer, David A Calderwood, Ankur Singh, David W Dumbauld, Susan W Craig, Emmanuel Delamarche, Andres J GarciaAbstract:Integrin-based focal adhesions (FA) transmit anchorage and traction forces between the cell and the extracellular matrix (ECM). To gain further insight into the physical parameters of the ECM that control FA assembly and force transduction in non-migrating cells, we used fibronectin (FN) nanopatterning within a cell adhesion-resistant background to establish the threshold area of ECM ligand required for stable FA assembly and force transduction. Integrin-FN clustering and adhesive force were strongly modulated by the geometry of the nanoscale adhesive area. Individual nanoisland area, not the number of nanoislands or total adhesive area, controlled Integrin-FN clustering and adhesion strength. Importantly, below an area threshold (0.11 µm(2)), very few Integrin-FN clusters and negligible adhesive forces were generated. We then asked whether this adhesive area threshold could be modulated by intracellular pathways known to influence either adhesive force, cytoskeletal tension, or the structural link between the two. Expression of talin- or vinculin-head domains that increase Integrin Activation or clustering overcame this nanolimit for stable Integrin-FN clustering and increased adhesive force. Inhibition of myosin contractility in cells expressing a vinculin mutant that enhances cytoskeleton-Integrin coupling also restored Integrin-FN clustering below the nanolimit. We conclude that the minimum area of Integrin-FN clusters required for stable assembly of nanoscale FA and adhesive force transduction is not a constant; rather it has a dynamic threshold that results from an equilibrium between pathways controlling adhesive force, cytoskeletal tension, and the structural linkage that transmits these forces, allowing the balance to be tipped by factors that regulate these mechanical parameters.
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structure of a double ubiquitin like domain in the talin head a role in Integrin Activation
2010Co-Authors: Benjamin T Goult, Neil Bate, Gordon C K Roberts, Bipin Patel, Alexandre R Gingras, David A Calderwood, Mohamed Bouaouina, Paul R Elliott, Gunter J Grossmann, David R CritchleyAbstract:Talin is a 270-kDa protein that activates Integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for β-Integrin tails, F0 and F1 are also required for Activation of β1-Integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated Activation of β1-Integrins. This and the fact that basic residues on F2 and F3 are also essential for Integrin Activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate Integrins.
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the structure of the n terminus of kindlin 1 a domain important for alphaiibbeta3 Integrin Activation
2009Co-Authors: Benjamin T Goult, Neil Bate, Igor L. Barsukov, Bipin Patel, Iain D. Campbell, David A Calderwood, Mohamed Bouaouina, David S Harburger, Nicholas J Anthis, Gordon C K RobertsAbstract:The Integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and Integrin Activation requires binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the β-Integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for talin-induced Integrin Activation. FERM domains are typically composed of F1, F2, and F3 domains, but the talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains. Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support talin-induced αIIbβ3 Integrin Activation and for the localization of kindlin-1 to focal adhesions.
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kindlin 1 and 2 directly bind the c terminal region of β Integrin cytoplasmic tails and exert Integrin specific Activation effects
2009Co-Authors: David S Harburger, Mohamed Bouaouina, David A CalderwoodAbstract:Integrin Activation, the rapid conversion of Integrin adhesion receptors from low to high affinity, occurs in response to intracellular signals that act on the short cytoplasmic tails of Integrin β subunits. Talin binding to Integrin β tails provides one key Activation signal, but additional factors are likely to cooperate with talin to regulate Integrin Activation. The Integrin β tail-binding proteins kindlin-2 and kindlin-3 were recently identified as Integrin co-activators. Here we report an analysis of kindlin-1 and kindlin-2 interactions with β1 and β3 Integrin tails and describe the effect of kindlin expression on Integrin Activation. We demonstrate a direct interaction of kindlin-1 and -2 with recombinant Integrin β tails in pulldown binding assays. Our mutational analysis shows that the second conserved NXXY motif (Tyr795), a preceding threonine-containing region (Thr788 and Thr789) of the Integrin β1A tail, and a conserved tryptophan in the F3 subdomain of the kindlin FERM domain (kindlin-1 Trp612 and kindlin-2 Trp615) are required for direct kindlin-Integrin interactions. Similar interactions were observed for Integrin β3 tails. Using fluorescence-activated cell sorting we further show that transient expression of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the Activation of endogenous α5β1 or stably expressed αIIbβ3 Integrins. This inhibition is not dependent on direct kindlin-Integrin interactions because mutant kindlins exhibiting impaired Integrin binding activity effectively inhibit Integrin Activation. Consistent with previous reports, we find that when co-expressed with the talin head, kindlin-1 or -2 can activate αIIbβ3. This effect is dependent on an intact Integrin-binding site in kindlin. Notably however, even when co-expressed with activating levels of talin head, neither kindlin-1 or -2 can cooperate with talin to activate β1 Integrins; instead they strongly inhibit talin-mediated Activation. We suggest that kindlins are adaptor proteins that regulate Integrin Activation, that kindlin expression levels determine their effects, and that kindlins may exert Integrin-specific effects.
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jam l mediated leukocyte adhesion to endothelial cells is regulated in cis by α4β1 Integrin Activation
2008Co-Authors: Annyclaude Luissint, David A Calderwood, P Lutz, Pierreolivier Couraud, Sandrine BourdoulousAbstract:Junctional adhesion molecules (JAMs) are endothelial and epithelial adhesion molecules involved in the recruitment of circulating leukocytes to inflammatory sites. We show here that JAM-L, a protein related to the JAM family, is restricted to leukocytes and promotes their adhesion to endothelial cells. Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor). Interestingly, JAM-L expressed on neutrophils binds CAR independently of Integrin Activation. However, on resting monocytes and T lymphocytes, which express the Integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form. Integrin Activation is required for the dissociation of JAM-L–VLA-4 complexes and the accumulation of functional JAM-L dimers, which indicates that the leukocyte Integrin VLA-4 controls JAM-L function in cis by controlling its dimerization state. This provides a mechanism through which VLA-4 and JAM-L functions are coordinately regulated, allowing JAM-L to strengthen Integrin-dependent adhesion of leukocytes to endothelial cells.
Klaus Ley - One of the best experts on this subject based on the ideXlab platform.
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rap1 binding and a lipid dependent helix in talin f1 domain promote Integrin Activation in tandem
2019Co-Authors: Alexandre R Gingras, Klaus Ley, Frederic Lagarrigue, Monica N Cuevas, Marcus Zorovich, Andrew J Valadez, Miguel Alejandro Lopezramirez, Nicolas Seban, Wilma Mclaughlin, William B KiossesAbstract:Rap1 GTPases bind effectors, such as RIAM, to enable talin1 to induce Integrin Activation. In addition, Rap1 binds directly to the talin1 F0 domain (F0); however, this interaction makes a limited contribution to Integrin Activation in CHO cells or platelets. Here, we show that talin1 F1 domain (F1) contains a previously undetected Rap1-binding site of similar affinity to that in F0. A structure-guided point mutant (R118E) in F1, which blocks Rap1 binding, abolishes the capacity of Rap1 to potentiate talin1-induced Integrin Activation. The capacity of F1 to mediate Rap1-dependent Integrin Activation depends on a unique loop in F1 that has a propensity to form a helix upon binding to membrane lipids. Basic membrane-facing residues of this helix are critical, as charge-reversal mutations led to dramatic suppression of talin1-dependent Activation. Thus, a novel Rap1-binding site and a transient lipid-dependent helix in F1 work in tandem to enable a direct Rap1-talin1 interaction to cause Integrin Activation.
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rap1 binding and a lipid dependent helix in talin f1 domain promote Integrin Activation in tandem
2019Co-Authors: Alexandre R Gingras, Klaus Ley, Frederic Lagarrigue, Monica N Cuevas, Marcus Zorovich, Andrew J Valadez, Miguel Alejandro Lopezramirez, Nicolas Seban, Wilma Mclaughlin, William B KiossesAbstract:Abstract Rap1 GTPases bind effectors, such as RIAM, to enable talin1 to induce Integrin Activation. In addition, Rap1 binds directly to the talin1 F0 domain (F0); however, this interaction makes a limited contribution to Integrin Activation in CHO cells or platelets. Here, we show that talin1 F1 domain contains a previously undetected Rap1 binding site of similar affinity to that in F0. A structure-guided point mutant (R118E) in F1, which blocks Rap1 binding, abolishes the capacity of Rap1 to potentiate talin1-induced Integrin Activation. The capacity of F1 to mediate Rap1-dependent Integrin Activation depends on a unique loop in F1 that transforms into an helix upon binding to membrane lipids. Basic membrane-facing residues of this helix are critical as charge reversal mutations led to dramatic suppression of talin1-dependent Activation. Thus, a novel Rap1 binding site and a lipid-dependent helix in talin1 F1 work in tandem to enable a direct Rap1-talin1 interaction to cause Integrin Activation. Summary This work reveals that Rap1 GTPases bind directly to talin1 F1 domain and by ooperating with a unique lipid-dependent amphipathic helix in the F1 domain effects lin1-mediated Integrin Activation.
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leukocyte arrest biomechanics and molecular mechanisms of β2 Integrin Activation
2016Co-Authors: Zhichao Fan, Klaus LeyAbstract:Integrins are a group of heterodimeric transmembrane receptors that play essential roles in cell-cell and cell-matrix interaction. Integrins are important in many physiological processes and diseases. Integrins acquire affinity to their ligand by undergoing molecular conformational changes called Activation. Here we review the molecular biomechanics during conformational changes of Integrins, Integrin functions in leukocyte biorheology (adhesive functions during rolling and arrest) and molecules involved in Integrin Activation.
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tyrosine kinase btk regulates e selectin mediated Integrin Activation and neutrophil recruitment by controlling phospholipase c plc γ2 and pi3kγ pathways
2010Co-Authors: Helena Mueller, Anika Stadtmann, Emilio Hirsch, Demin Wang, Alexander Zarbock, Klaus Ley, Hugo Van AkenAbstract:Selectins mediate leukocyte rolling, trigger β2-Integrin Activation, and promote leukocyte recruitment into inflamed tissue. E-selectin binding to P-selectin glycoprotein ligand 1 (PSGL-1) leads to Activation of an immunoreceptor tyrosine-based Activation motif (ITAM)–dependent pathway, which in turn activates the spleen tyrosine kinase (Syk). However, the signaling pathway linking Syk to Integrin Activation after E-selectin engagement is unknown. To identify the pathway, we used different gene-deficient mice in autoperfused flow chamber, intravital microscopy, peritonitis, and biochemical studies. We report here that the signaling pathway downstream of Syk divides into a phospholipase C (PLC) γ2– and phosphoinositide 3-kinase (PI3K) γ–dependent pathway. The Tec family kinase Bruton tyrosine kinase (Btk) is required for activating both pathways, generating inositol-3,4,5-trisphosphate (IP3), and inducing E-selectin–mediated slow rolling. Inhibition of this signal-transduction pathway diminished Gαi-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster. Gαi-independent neutrophil recruitment into the inflamed peritoneal cavity was reduced in Btk−/− and Plcg2−/− mice. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by E-selectin engagement.
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cells on the run shear regulated Integrin Activation in leukocyte rolling and arrest on endothelial cells
2008Co-Authors: Ronen Alon, Klaus LeyAbstract:The arrest of rolling leukocytes on various target vascular beds is mediated by specialized leukocyte Integrins and their endothelial immunoglobulin superfamily (IgSF) ligands. These Integrins are kept in largely inactive states and undergo in situ Activation upon leukocyte-endothelial contact by both biochemical and mechanical signals from flow-derived shear forces. In vivo and in vitro studies suggest that leukocyte Integrin Activation involves conformational alterations through inside-out signaling followed by ligand-induced rearrangements accelerated by external forces. This Activation process takes place within fractions of seconds by in situ signals transduced to the rolling leukocyte as it encounters specialized endothelial-displayed chemoattractants, collectively termed arrest chemokines. In neutrophils, selectin rolling engagements trigger intermediate affinity Integrins to support reversible adhesions before chemokine-triggered arrest. Different leukocyte subsets appear to use different modalities of Integrin Activation during rolling and arrest at distinct endothelial sites.