New perspectives on integrin-dependent adhesions

Integrins are heterodimeric transmembrane receptors that connect the extracellular matrix environment to the actin cytoskeleton via adaptor molecules through assembly of a range of adhesion structures. Recent advances in biochemical, imaging and biophysical methods have enabled a deeper understanding of integrin signalling and their associated regulatory processes. The identification of the consensus integrin-based ‘adhesomes’ within the last 5 years has defined common core components of adhesion complexes and associated partners. These approaches have also uncovered unexpected adhesion protein behaviour and molecules recruited to adhesion sites that have expanded our understanding of the molecular and physical control of integrin signalling.     

Integrin αIIbβ3 in a Membrane Environment Remains the Same Height after Mn Activation when Observed by Cryoelectron Tomography

Integrins perform the critical function of signalling cell attachment to the extracellular matrix or to other cells. This signalling is done through a structural change propagated bidirectionally across the plasma membrane. Integrin activation has been extensively studied with ectodomain constructs, but the structural change within intact, membrane-bound molecules remains a subject of live debate. Using cryoelectron tomography, we examined the simplest predication of the different integrin activation models, i.e., the change in height of the molecules. Analysis using techniques that compensate for the missing wedge during alignment and averaging and that search for patterns in the structure of the aligned molecular subvolumes extracted from the tomogram reveals that the vast majority of molecules show no dramatic height change upon Mn²⁺-induced activation of membrane-bound integrins when compared with an inactive integrin control group. Thus, the result is inconsistent with the switchblade activation model.     

整合素的活化调控

整合素家族是介导细胞与细胞外基质作用的最主要分子,不仅可以识别细胞外环境将信号传到细胞内,还可以通过来自细胞内的信号调节整合素和配体的亲和力,这个过程也就是整合素的活化。本文主要阐述了整合素的活化在生理过程中的重要作用、整合素活性调节的结构基础以及细胞内的信号通路和结合蛋白对整合素活性的影响。     

Integrin-collagen complex: a metal-glutamate handshake

The recently determined crystal structure of the complex between an integrin I domain and a synthetic collagen peptide shows a collagen triple-helix engaged in specific macromolecular recognition and binding. This structure confirms a previously proposed binding mechanism for integrin I domains and has important implications for integrin activation and signalling.     

Cα-H···O=C hydrogen bonds contribute to the specificity of RGD cell-adhesion interactions

Background  

The Arg-Gly-Asp (RGD) cell adhesion sequence occurs in several extracellular matrix molecules known to interact with integrin cell-surface receptors. Recently published crystal structures of the extracellular regions of two integrins in complex with peptides containing or mimicking the RGD sequence have identified the Arg and Asp residues as key specificity determinants for integrin recognition, through hydrogen bonding and metal coordination interactions. The central Gly residue also appears to be in close contact with the integrin surface in these structures.     

Integrins: versatile integrators of extracellular signals

Extracellular matrix (ECM) molecules and growth factors have a crucial role in the signalling that controls cell behaviour during development. Integrins, which are cell-surface receptors for ECM molecules, and growth factor receptors cooperate with each other to regulate this signalling by several mechanisms. In particular, direct interactions between the integrin and growth factor receptors themselves, which often occur within a single macromolecular complex, amplify signalling by mechanisms that include posttranslational modifications and integrin shape changes that are related to activation. As a result, growth factor concentrations in the physiological range, which are too low to initiate signalling alone, do so in the presence of the ECM, enabling integrins to control the time and space of growth factor signalling.     

The structure of the integrin αIIbβ3 transmembrane complex explains integrin transmembrane signalling

Heterodimeric integrin adhesion receptors regulate cell migration, survival and differentiation in metazoa by communicating signals bi‐directionally across the plasma membrane. Protein engineering and mutagenesis studies have suggested that the dissociation of a complex formed by the single‐pass transmembrane (TM) segments of the α and β subunits is central to these signalling events. Here, we report the structure of the integrin αIIbβ3 TM complex, structure‐based site‐directed mutagenesis and lipid embedding estimates to reveal the structural event that underlies the transition from associated to dissociated states, that is, TM signalling. The complex is stabilized by glycine‐packing mediated TM helix crossing within the extracellular membrane leaflet, and by unique hydrophobic and electrostatic bridges in the intracellular leaflet that mediate an unusual, asymmetric association of the 24‐ and 29‐residue αIIb and β3 TM helices. The structurally unique, highly conserved integrin αIIbβ3 TM complex rationalizes bi‐directional signalling and represents the first structure of a heterodimeric TM receptor complex.     

Signalling via integrins: implications for cell survival and anticancer strategies

Integrin-associated signalling renders cells more resistant to genotoxic anti-cancer agents like ionizing radiation and chemotherapeutic substances, a phenomenon termed cell adhesion-mediated radioresistance/drug resistance (CAM-RR, CAM-DR). Integrins are heterodimeric cell-surface molecules that on one side link the actin cytoskeleton to the cell membrane and on the other side mediate cell-matrix interactions. In addition to their structural functions, integrins mediate signalling from the extracellular space into the cell through integrin-associated signalling and adaptor molecules such as FAK (focal adhesion kinase), ILK (integrin-linked kinase), PINCH (particularly interesting new cysteine-histidine rich protein) and Nck2 (non-catalytic (region of) tyrosine kinase adaptor protein 2). Via these molecules, integrin signalling tightly and cooperatively interacts with receptor tyrosine kinase signalling to regulate survival, proliferation and cell shape as well as polarity, adhesion, migration and differentiation. In tumour cells of diverse origin like breast, colon or skin, the function and regulation of these molecules is partly disturbed and thus might contribute to the malignant phenotype and pre-existent and acquired multidrug resistance. These issues as well as a variety of therapeutic options envisioned to influence tumour cell growth, metastasis and resistance, including kinase inhibitors, anti-integrin antibodies or RNA interference, will be summarized and discussed in this review.     

Integrin regulation of caveolin function

Caveolae are unique organelles that are found in the plasma membrane of many cell types. They participate in various processes such as lipid recycling, cellular signalling and endocytosis. A variety of signalling molecules localize to caveolae in response to various stimuli, providing a potential mechanism for the spatial regulation of signal transduction pathways. Caveolin-1, a constitutive protein of caveolae, has been implicated in the regulation of cell growth, lipid trafficking, endocytosis and cell migration. Phosphorylation of caveolin-1 on Tyr 14 is involved in integrin-regulated caveolae trafficking and also in signalling at focal adhesions in migrating cells. In this review, we focus on recent studies that describe the role of caveolin-1 in integrin signal transduction, and how this interplay links extracellular matrix anchorage to cell proliferation, polarity and directional migration.     

Structural and mechanical functions of integrins

Integrins are ubiquitously expressed cell surface receptors that play a critical role in regulating the interaction between a cell and its microenvironment to control cell fate. These molecules are regulated either via their expression on the cell surface or through a unique bidirectional signalling mechanism. However, integrins are just the tip of the adhesome iceberg, initiating the assembly of a large range of adaptor and signalling proteins that mediate the structural and signalling functions of integrin. In this review, we summarise the structure of integrins and mechanisms by which integrin activation is controlled. The different adhesion structures formed by integrins are discussed, as well as the mechanical and structural roles integrins play during cell migration. As the function of integrin signalling can be quite varied based on cell type and context, an in depth understanding of these processes will aid our understanding of aberrant adhesion and migration, which is often associated with human pathologies such as cancer.     

Protein tyrosine phosphorylation and the adhesive functions of platelets

The intracellular signalling pathways that mediate changes in cell behavior induced by extracellular matrix and cell adhesion molecules are poorly understood. Studies on the regulation of tyrosine phosphorylation in platelets indicate that cell-to-cell aggregation mediated by fibrinogen binding to its integrin-family receptor, GP IIb-IIIa, and events regulated by the putative adhesion receptor, GP IV (CD36), involve tyrosine phosphorylation. Thus, tyrosine phosphorylation is implicated in cellular events crucial for hemostasis. It may also be involved in signaling mediated by integrin receptors in other cell types.     

A naturally occurring extracellular alpha-beta clasp contributes to stabilization of beta3 integrins in a bent, resting conformation

Control of alphaIIb beta3 and alphav beta3 integrin activation is critical for cardiovascular homeostasis. Mutations that perturb association of integrin alpha and beta subunits in their transmembrane and cytoplasmic regions activate the integrin heterodimer, suggesting that a low-affinity or "off" conformation is the default state, likely corresponding to the bent conformation seen in the crystal structure of alphav beta3. In this bent structure, a segment of alphav (301-308) and beta3 (560-567) are juxtaposed. Here we provide evidence that these regions of alphav/alphaIIb and beta3 function as a novel extracellular clasp to restrain activation. Synthetic peptides representing the alphaIIb and beta3 clasp regions promote integrin activation as judged by cell adhesion, cell spreading, and exposure of epitopes for three beta3 LIBS antibodies. Mutation of the clasp region of alphav or beta3 results in a constitutively activated integrin, confirming the role of the extracellular clasp in restraining integrin activation. Molecular dynamics simulations of the alphav beta3 structure yield a refined model for the alphav beta3 clasp and provide plausible explanations for the effects of the activating mutations.     

The structure of an integrin/talin complex reveals the basis of inside‐out signal transduction

Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that uniquely mediate inside‐out signal transduction, whereby adhesion to the extracellular matrix is activated from within the cell by direct binding of talin to the cytoplasmic tail of the β integrin subunit. Here, we report the first structure of talin bound to an authentic full‐length β integrin tail. Using biophysical and whole cell measurements, we show that a specific ionic interaction between the talin F3 domain and the membrane–proximal helix of the β tail disrupts an integrin α/β salt bridge that helps maintain the integrin inactive state. Second, we identify a positively charged surface on the talin F2 domain that precisely orients talin to disrupt the heterodimeric integrin transmembrane (TM) complex. These results show key structural features that explain the ability of talin to mediate inside‐out TM signalling.     

Ras GTPases: integrins' friends or foes?

Integrins are cell-surface receptors that mediate and coordinate cellular responses to the extracellular matrix (ECM). Cellular signalling pathways can regulate cell adhesion by altering the affinity and avidity of integrins for ECM. The Ras family of small G proteins, which includes H-ras, R-ras and Rap, are important elements in cellular signalling pathways that control integrin function.     

Down-Regulation of Integrin β1 and Focal Adhesion Kinase in Renal Glomeruli under Various Hemodynamic Conditions

Given that integrin β1 is an important component of the connection to maintain glomerular structural integrity, by binding with multiple extracellular matrix proteins and mediating intracellular signaling. Focal adhesion kinase (FAK) is the most essential intracellular integrator in the integrin β1-FAK signalling pathway. Here, we investigated the changes of the two molecules and visualized the possbile interaction between them under various hemodynamic conditions in podocytes. Mice kidney tissues were prepared using in vivo cryotechnique (IVCT) and then were stained and observed using light microscopy, confocal laser scanning microscopy and immunoelectron microscopy. The expression of these molecules were examined by western blot. Under the normal condition, integrin β1 stained continually and evenly at the membrane, and FAK was located in the cytoplasm and nuclei of the podocytes. There were significant colocalized plaques of two molecules. But under acute hypertensive and cardiac arrest conditions, integrin β1 decreased and stained intermittently. Similarly, FAK decreased and appeared uneven. Additionally, FAK translocated to the nuclei of the podocytes. As a result, the colocalization of integrin β1 and FAK reduced obviously under these conditions. Western blot assay showed a consistent result with the immunostaining. Collectively, the abnormal redistribution and decreased expressions of integrin β1 and FAK are important molecular events in regulating the functions of podocytes under abnormal hemodynamic conditions. IVCT could offer considerable advantages for morphological analysis when researching renal diseases.     

Tensins are versatile regulators of Rho GTPase signalling and cell adhesion

Tensins are focal adhesion molecules that were identified and characterised in the late 1980s to early 1990s. They play an essential role in the control of cell adhesion. Tensins can bind the tail of ß integrin via their phospho tyrosine binding domain, they exhibit various protein interaction domains including a Src Homology 2 domain and they are serine‐, threonine‐ and tyrosine‐phosphorylated in response to various stimuli. Tensins serve as scaffolds to gather signalling molecules at the extracellular matrix adhesion complexes. Tensins have emerged as important regulators of cell adhesion and migration, in particular by participating in Rho GTPase signalling pathways. Tensins were shown to influence the activity of the GTPase RhoA, by regulating the Rho GTPase activating protein Deleted in Liver Cancer 1. More recently, Tensin 3 was also found to regulate Dock5, a guanine nucleotide exchange factor for the GTPase Rac, and to modulate podosome‐based adhesion structures in osteoclasts. This review focusses on the recent literature highlighting how Tensins can interplay with regulators of Rho GTPase signalling pathways and how this influences cell adhesion and migration.     

Integrin Signaling: Cytoskeletal Complexes,MAP Kinase Activation,and Regulation of Gene Expression

Members of the integrin family of adhesion receptors mediate interactions of cells with the extracellular matrix. Besides their role in tissue morphogenesis by anchorage of cells to basement membranes and migration along extracellular matrix proteins, integrins are thought to play a key role in mediating the control of gene expression by the extracellular matrix. Studies over the past 10 years have shown that integrin-mediated cell adhesion can trigger signal transduction cascades involving translocation of proteins and protein tyrosine phosphorylation events. In this review, we discuss approaches used in our lab to study early events in integrin signalling as well as further downstream changes.     

Integrin-linked kinase (ILK): a regulator of integrin and growth-factor signalling.

Interaction of cells with the extracellular matrix (ECM) results in the regulation of cell growth, differentiation and migration by coordinated signal transduction through integrins and growth-factor receptors. Integrins achieve signalling by interacting with intracellular effectors that couple integrins and growth-factor receptors to downstream components. One well-studied effector is focal-adhesion kinase (FAK), but recently another protein kinase, integrin-linked kinase (ILK), has been identified as a receptor-proximal effector of integrin and growth-factor signalling. ILK appears to interact with and be influenced by a number of different signalling pathways, and this provides new routes for integrin-mediated signalling. This article discusses ILK structure and function and recent genetic and biochemical evidence about the role of ILK in signal transduction.     

Cysteine-rich module structure reveals a fulcrum for integrin rearrangement upon activation

Cysteine-rich repeats in the integrin beta subunit stalk region relay activation signals to the ligand-binding headpiece. The NMR solution structure and disulfide bond connectivity of Cys-rich module-3 of the integrin beta2 subunit reveal a nosecone-shaped variant of the EGF fold, termed an integrin-EGF (I-EGF) domain. Interdomain contacts between I-EGF domains 2 and 3 observed by NMR support a model in which the modules are related by an approximate two-fold screw axis in an extended arrangement. Our findings complement a 3.1 A crystal structure of the extracellular portion of integrin alphaVbeta3, which lacks an atomic model for I-EGF2 and a portion of I-EGF3. The disulfide connectivity of I-EGF3 chemically assigned here differs from the pairings suggested in the alphaVbeta3 structure. Epitopes that become exposed upon integrin activation and residues that restrain activation are defined in beta2 I-EGF domains 2 and 3. Superposition on the alphaVbeta3 structure reveals that they are buried. This observation suggests that the highly bent alphaVbeta3 structure represents the inactive conformation and that release of contacts with I-EGF modules 2 and 3 triggers a switchblade-like opening motion extending the integrin into its active conformation.