Talin – the master of integrin adhesions
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ABSTRACT Talin has emerged as the key cytoplasmic protein that mediates integrin adhesion to the extracellular matrix. In this Review, we draw on experiments performed in mammalian cells in culture and Drosophila to present evidence that talin is the most important component of integrin adhesion complexes. We describe how the properties of this adaptor protein enable it to orchestrate integrin adhesions. Talin forms the core of integrin adhesion complexes by linking integrins directly to actin, increasing the affinity of integrin for ligands (integrin activation) and recruiting numerous proteins. It regulates the strength of integrin adhesion, senses matrix rigidity, increases focal adhesion size in response to force and serves as a platform for the building of the adhesion structure. Finally, the mechano-sensitive structure of talin provides a paradigm for how proteins transduce mechanical signals to chemical signals.Keywords:
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사람과 동일한 증상을 나타내는 동물 모델이 없는 신증후출혈열의 병리 기전을 이해하기 위하여 in vitro culture system을 이용하였다. 신증후출혈열의 주된 병변이 혈관내피세포외에 섬유아세포가 많이 존재하는 신세뇨관 간질 및 폐간질임을 기초로 하여 계태아와 Mongolian gerbil의 섬유아 세포에서 fibronectin에 대한 수용체인 a 5 β 1 ， integrin에 대한 항체를 처리하여 한탄바이러스 감염에 대한 영향을 관찰하였다. 초대 배양하여 사용한 계태아 섬유아세포 및 MGF 모두 한탄바이러스가 잘 감염되었으나 계태아 섬유아세포에 비하여 MGF에서 바이러스의 증식이 더 우수하였다. 계태아 섬유아세포와 MGF에서 a 5 β 1 , integrin에 대한 항체 처리 5일 후 바이러스의 N 단백의 역가는 항체를 처리하지 않은 대조군에 비하여 각각 32.6%, 72.6%로 virion의 역가는 26.8%, 28.7%로 감소하여 두 세포에서 모두 감염력을 가진 바이러스의 양은 거의 동일하게 감소 되었다. 또한 a 5 subunit와 s 1 , subunit에 대한 항체를 MGF 세포에 처리한 후 바이러스의 N단백의 역가는 65.2%, 59.7%로 a 5 β 1 integrin에 대한 항체 처리하였을 때 (72.6%)와 거의 유사하게 감소하였고 감염력을 가진 바이러스의 양도 26.5%, 29.4%로 감소하여 a 5 β 1 integrin에 대한 항체 처리시 (28.7%)와 거의 유사하게 감소하였다. 이러한 결과는 a 5 β 1 integrin이 한탄바이러스에 대한 수용체로써 작용할 가능성을 시사하였으며 또 다른 기전으로는 a 5 β 1 integrin의 차단에 의한 이차적인 영향으로 한탄바이러스의 증식에 커다란 영향을 미치는 것을 생각할 수 있었다.
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Integrins are heterodimeric trans-membrane receptor proteins that mediate the interaction of cells with extracellular matrix proteins (ECM). They mediate various growth factor dependent cell-signaling pathways during the development of fibrosis that is characteristic of all forms of chronic kidney diseases. Although integrin β1 is the most abundant integrin subunit in kidney and can form complexes with 12 different α subunits, integrin β3 is the best studied integrin β subunit and serves as the canonical model for integrin function based on the high sequence homology between the trans-membrane and cytosolic domains of integrin β subunits. A conserved lysine residue towards the C-terminus of integrin β3 subunit is reported to be important for regulating the activation of integrin αIIbβ3 complexes; however the functional importance of this lysine is unknown in β1 integrins. We investigated the role of this lysine residue in integrin β1-dependent kidney collecting duct cell function. We expressed the mutant protein where the lysine is mutated to glutamic acid in collecting duct cells null for integrin β1. Collecting duct cells expressing mutant protein had decreased the adhesion of cells to collagen IV mediated by integrin α1β1 by 80% (0.95 vs 0.18). This mutation also decreased the ability of IMCD cells adhesion to collagen I mediated by integrin α2β1 by 82% (0.78 vs 0.15). In contrast to earlier reports in integrin β3, this mutation did not significantly alter the amount of active integrin β1 on the cell surface as estimated by FACS analysis; however we did observe a decrease in conformation specific antibody binding on cells adhered to collagen (0.70 vs 0.30). We also investigated the role of this lysine residue in complex formation of purified integrin β1 with integrin α1 and α2 TM/CT domains in phospholipid bicelles using fluorescence anisotropy. The dissociation constant for binding was estimated to be >3.2 mol and mutation of lysine residue did not significantly alter their binding ability. This contrasted with integrin αIIb β3 where we found fourfold decrease in binding ability (Kd 0.09 ± 0.03 mol and 0.33 ± 0.05 mol). Our data clearly suggest that conserved transmembrane lysine in both integrin β3 and integrin β1 regulate cell functions by distinct mechanisms.
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The integrin β1 subunit cytoplasmic tail forms oligomers: a potential role in β1 integrin clustering
Abstract Integrins are α/β heterodimeric cell surface receptors devoid of enzymatic activity. Signal transduction therefore requires the association of cytosolic and cytoskeletal proteins with the integrin subunit intracellular regions. This association is initiated upon ligand binding to the integrin receptor and includes clustering of the integrins and recruitment of focal adhesion‐associated proteins. Whether integrin clustering is solely dependent on ligand binding to the integrin extracellular parts or involves also interactions between the intracellular tails of integrins is so far unknown. To investigate intracellular events in integrin clustering, we have used peptides corresponding to the integrin β1 cytoplasmic region. Loading of cells with the peptides results in a decreased cell adhesion and in an inhibition of cell spreading in agreement with the previously reported dominant negative effect of the β1 integrin cytoplasmic tail on integrin clustering. Direct protein—protein interaction studies by surface plasmon resonance demonstrate that integrin β1 cytoplasmic peptides self‐associate in contrast to integrin β3 cytoplasmic tails. Size exclusion chromatography and SDS—PAGE analysis of the peptides further show that the integrin β1 cytoplasmic parts form oligomers and that they assume α helical conformation to the extent of about 13% and that this fraction is increased upon aggregation. Thus self‐association of the integrin β1 subunit cytoplasmic regions may be central to β1 integrin clustering.
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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 beta subunits. Talin binding to integrin beta tails provides one key activation signal, but additional factors are likely to cooperate with talin to regulate integrin activation. The integrin beta 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 beta1 and beta3 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 beta tails in pulldown binding assays. Our mutational analysis shows that the second conserved NXXY motif (Tyr(795)), a preceding threonine-containing region (Thr(788) and Thr(789)) of the integrin beta1A tail, and a conserved tryptophan in the F3 subdomain of the kindlin FERM domain (kindlin-1 Trp(612) and kindlin-2 Trp(615)) are required for direct kindlin-integrin interactions. Similar interactions were observed for integrin beta3 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 alpha5beta1 or stably expressed alphaIIbbeta3 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 alphaIIbbeta3. 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 beta1 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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Control of integrin affinity for ligands (integrin activation) is essential for normal cell adhesion, migration, and assembly of an extracellular matrix. Integrin activation is usually mediated through the integrin beta subunit cytoplasmic tail and can be regulated by many different biochemical signaling pathways. We report that specific binding of the cytoskeletal protein talin to integrin beta subunit cytoplasmic tails leads to the conformational rearrangements of integrin extracellular domains that increase their affinity. Thus, regulated binding of talin to integrin beta tails is a final common element of cellular signaling cascades that control integrin activation.
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In recent years, our understanding of the molecular interaction of collagens with their cognate integrin receptors has remarkably improved. Structural elucidations of both the integrin and the collagenous triple helix have contributed to this achievement. The structures of an entire integrin ectodomain and of an A-domain, which is unique to the integrin α subunits of collagen-binding and leukocyte integrins, have been resolved crystallographically. Furthermore, a complex of such an integrin α subunit A-domain with its collagenous binding partner has revealed their interaction on the molecular level and gave first evidence in the conformational alterations which may convey the signal of ligand occupancy through the integrin into the cells. In parallel, the tissue distribution and biological functions of collagen-binding integrins have been characterised. Nowadays, the contribution of distinct integrins to different physiological and pathological processes is known. Among the best studied examples is the collagen-induced platelet activation and aggregation, in which α2β1 integrin is involved. Together with α1β1 integrin, it also plays a role in inflammatory processes. To manipulate processes which are mediated by collagen-binding integrins, compounds are developed which mimic the collagen ligand. Not only the structural information of the integrin:collagen-interaction but also improvements in the chemical synthesis of a collagenous triple helix facilitate the development of agonists and antagonists of collagen-binding integrins. Furthermore, another impact in this search comes from the discovery of high-affinity inhibitors from venoms, which lack a collagenous triple-helix. Keywords: integrin, collagen, disintegrin, integrin agonist, integrin antagonist, cell-matrix interaction
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We report data showing that the integrin receptor complex in chickens contains several discrete heterodimers all sharing the beta 1-integrin subunit combined separately with different alpha-subunits. Using antisera to synthetic peptides based on cDNA sequences of chicken and human alpha-integrin subunits to analyze the integrin complement of avian and mammalian cells, we show that band 2 of the chicken integrin complex contains alpha-subunits related to both alpha 3- and alpha 5-subunits of human integrins. alpha 3 beta 1 and alpha 5 beta 1 have both previously been shown in human cells to be fibronectin receptors and alpha 3 beta 1 can also act as a receptor for laminin and collagen. We also provide evidence for the presence, in band 1 of the chicken integrin complex, of a third integrin alpha-subunit which is also alpha 5 related. This integrin subunit exists in a separate heterodimer complex with beta 1 and binds to fibronectin-affinity columns. These results provide explanations for published data showing that the avian integrin complex contains receptor activity for a variety of extracellular matrix proteins. We conclude that the chicken integrin complex comprises a set of beta 1-integrin heterodimers equivalent to the human VLA antigens and includes at least two fibronectin receptors. Finally, we show that chicken embryo fibroblasts also contain a beta 3-class integrin related to the RGD receptors defined in various human cells.
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