Susan E. Craig

Structure of an Integrin-Ligand Complex Deduced from Solution X-ray Scattering and Site-directed Mutagenesis

The structural basis of the interaction of integrin heterodimers with their physiological ligands is poorly understood. We have used solution x-ray scattering to visualize the head region of integrin α5β1 in an inactive (Ca2+-occupied) state, and in complex with a fragment of fibronectin containing the RGD and synergy recognition sequences. Shape reconstructions of the data have been interpreted in terms of appropriate molecular models. The scattering data suggest that the head region undergoes no gross conformational changes upon ligand binding but do lend support to a proposed outward movement of the hybrid domain in the β subunit. Fibronectin is observed to bind across the top of the head region, which contains an α subunit β-propeller and a β subunit vWF type A domain. The model of the complex indicates that the synergy region binds on the side of the β-propeller domain. In support of this suggestion, mutagenesis of a prominent loop region on the side of the propeller identifies two residues (Tyr208 and Ile210) involved in recognition of the synergy region. Our data provide the first view of a complex between an integrin and a macromolecular ligand in solution, at a nominal resolution of ∼10 Å.

A specific α5β1-integrin conformation promotes directional integrin translocation and fibronectin matrix formation

Integrin adhesion receptors are structurally dynamic proteins that adopt a number of functionally relevant conformations. We have produced a conformation-dependent anti-α5 monoclonal antibody (SNAKA51) that converts α5β1 integrin into a ligand-competent form and promotes fibronectin binding. In adherent fibroblasts, SNAKA51 preferentially bound to integrins in fibrillar adhesions. Clustering of integrins expressing this activation epitope induced directional translocation of α5β1, mimicking fibrillar adhesion formation. Priming of α5β1 integrin by SNAKA51 increased the accumulation of detergent-resistant fibronectin in the extracellular matrix, thus identifying an integrin conformation that promotes matrix assembly. The SNAKA51 epitope was mapped to the calf-1/calf-2 domains. We propose that the action of the antibody causes the legs of the integrin to change conformation and thereby primes the integrin to bind ligand. These findings identify SNAKA51 as the first anti-integrin antibody to selectively recognize a subset of adhesion contacts, and they identify an integrin conformation associated with integrin translocation and fibronectin matrix formation.

Role of ADMIDAS Cation-binding Site in Ligand Recognition by Integrin α5β1

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and multiple cation-binding sites are found in both α and β integrin subunits. A key cation-binding site that lies in the β subunit A-domain is known as the metal-ion dependent adhesion site (MIDAS). Recent x-ray crystal structures of integrin αVβ3 have identified a novel cation binding site in this domain, known as the ADMIDAS (adjacent to MIDAS). The role of this novel site in ligand recognition has yet to be elucidated. Using the interaction between α5β1 and fibronectin as a model system, we show that mutation of residues that form the ADMIDAS site inhibits ligand binding but this effect can be partially rescued by the use of activating monoclonal antibodies. The ADMIDAS mutants had decreased expression of activation epitopes recognized by 12G10, 15/7, and HUTS-4, suggesting that the ADMIDAS is important for stabilizing the active conformation of the integrin. Consistent with this suggestion, the ADMIDAS mutations markedly increased the dissociation rate of the integrin-fibronectin complex. Mutation of the ADMIDAS residues also reduced the allosteric inhibition of Mn2+-supported ligand binding by Ca2+, suggesting that the ADMIDAS is a Ca2+-binding site involved in the inhibition of Mn2+-supported ligand binding. Mutations of the ADMIDAS site also perturbed transduction of a conformational change from the MIDAS through the C-terminal helix region of the βA domain to the underlying hybrid domain, implying an important role for this site in receptor signaling.

Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins

Significance Mechanical forces, which guide cellular functions, can be sensed and translated into biochemical information at focal adhesions, where cells physically connect to extracellular matrix (ECM) through transmembrane receptor integrins. Our results have identified that different ECM proteins, type 1 collagen (Col I) and fibronectin (FN), can either transmit or shield from mechanical forces when regulating crucial intracellular signaling, focal adhesion kinase (FAK), with their different accessibility to the corresponding integrin receptors. Whereas the integrin α2 binding site in Col I is constitutively accessible, mechanical tension is required to expose the integrin α5 binding motif in FN. This finding should advance our understanding on how cells perceive extracellular mechanical cues through natural surface materials. Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin β1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.

E-cadherin is a ligand for integrin α2β1

Abstract E-cadherin is a 120-kDa transmembrane glycoprotein expressed mainly on the surface of epithelial cells. The best characterised function of E-cadherin is homotypic, calcium-dependent cell–cell adhesion; however, the observation that E-cadherin is also capable of interacting with the αEβ7 integrin to mediate leukocyte cell–cell adhesion [Nature 372 (1994) 190] suggests that it also participates in heterotypic interactions. To investigate the possibility that E-cadherin may interact with integrins expressed on non-leukocytic cells, cell adhesion and solid-phase receptor–ligand binding experiments were performed using a pentameric E-cadherin construct designed to detect low affinity, high avidity interactions. HT1080 human fibrosarcoma cells specifically adhered to pentameric E-cadherin, and this adhesion was inhibited by anti-functional monoclonal antibodies directed against the integrin α2 and β1 subunits, but not by a series of antibodies recognising other subunits. This suggested that the E-cadherin receptor was α2β1, a previously characterised collagen/laminin receptor. Pentameric E-cadherin, but not monomeric E-cadherin, specifically bound, in a divalent cation-dependent manner, to both purified α2β1 and to a recombinant form of the A-domain of the α2 subunit, which has been shown to be a major ligand-binding site within this and other integrins. These findings demonstrate that E-cadherin can interact with α2β1 and suggest that heterotypic interactions between E-cadherin and integrins may be more common than originally thought.

Activation of integrin α5β1 delays apoptosis of Ntera2 neuronal cells

Integrins are dynamic membrane proteins that mediate adhesion of cells to the extracellular matrix. Integrins initiate signal transduction, alone and cooperatively with growth factor receptors, and regulate many aspects of cell behavior. We report here that α5β1-mediated adhesion of Ntera2 neuronal cells to fibronectin decreased apoptosis in response to serum withdrawal. Adhesion induced phosphorylation of FAK, and strongly increased the AKT phosphorylation induced by growth factors, demonstrating for the first time in neuronal cells that integrin-mediated adhesion and growth factors cooperate to regulate AKT activity. Integrins exist on cells in different activation states, and cell survival on fibronectin was enhanced by the antibody 12G10, that modulates the conformation of β1 in favor of its active form. The antibody 12G10 specifically delayed loss of phosphorylation of AKT on serine 473, and GSK-3β on serine 9, induced by serum withdrawal, suggesting that these kinases are critical sensors of integrin activation on neuronal cells.

Fibronectin supports neurite outgrowth and axonal regeneration of adult brain neurons in vitro.

The molecular basis of axonal regeneration of central nervous system (CNS) neurons remains to be fully elucidated. In part, this is due to the difficulty in maintaining CNS neurons in vitro. Here, we show that dissociated neurons from the cerebral cortex and hippocampus of adult mice may be maintained in culture for up to 9 days in defined medium without added growth factors. Outgrowth of neurites including axons was observed from both CNS sources and was significantly greater on plasma fibronectin than on other substrata such as laminin and merosin. Neurite outgrowth on fibronectin appears to be mediated by α5β1 integrin since a recombinant fibronectin fragment containing binding sites for this receptor was as effective as intact fibronectin in supporting neurite outgrowth. Conversely, function-blocking antibodies to α5 and β1 integrin sub-units inhibited neurite outgrowth on intact fibronectin. These results suggest that the axonal regeneration seen in in vivo studies using fibronectin-based matrices is due to the molecule itself and not a consequence of secondary events such as cellular infiltration. They also indicate the domains of fibronectin that may be responsible for eliciting this response.

Novel activating and inactivating mutations in the integrin β1 subunit A domain

The ligand-binding activity of integrins is regulated by shape changes that convert these receptors from a resting (or inactive) state to an active state. However, the precise conformational changes that take place in head region of integrins (the site of ligand binding) during activation are not well understood. The portion of the integrin beta subunit involved in ligand recognition contains a von Willebrand factor type A domain, which comprises a central beta-sheet surrounded by seven alpha helices (alpha1-alpha7). Using site-directed mutagenesis, we show here that point mutation of hydrophobic residues in the alpha1 and alpha7 helices (which would be predicted to increase the mobility of these helices) markedly increases the ligand-binding activity of both integrins alpha5beta1 and alpha4beta1. In contrast, mutation of a hydrophilic residue near the base of the alpha1 helix decreases activity and also suppresses exposure of activation epitopes on the underlying hybrid domain. Our results provide new evidence that shifts of the alpha1 and alpha7 helices are involved in activation of the A domain. Although these changes are grossly similar to those defined in the A domains found in some integrin alpha subunits, movement of the alpha1 helix appears to play a more prominent role in betaA domain activation.