A. Paul Mould

Demonstration of catch bonds between an integrin and its ligand

Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules. How force regulates integrin–ligand dissociation is unclear. We used atomic force microscopy to measure the force-dependent lifetimes of single bonds between a fibronectin fragment and an integrin α5β1-Fc fusion protein or membrane α5β1. Force prolonged bond lifetimes in the 10–30-pN range, a counterintuitive behavior called catch bonds. Changing cations from Ca2+/Mg2+ to Mg2+/EGTA and to Mn2+ caused longer lifetime in the same 10–30-pN catch bond region. A truncated α5β1 construct containing the headpiece but not the legs formed longer-lived catch bonds that were not affected by cation changes at forces <30 pN. Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range. Thus, catch bond formation appears to involve force-assisted activation of the headpiece but not integrin extension.

Linking integrin conformation to function

Integrins are αβ heterodimeric adhesion receptors that relay signals bidirectionally across the plasma membrane between the extracellular matrix and cell-surface ligands, and cytoskeletal and signalling effectors. The physical and chemical signals that are controlled by integrins are essential for intercellular communication and underpin all aspects of metazoan existence. To mediate such diverse functions, integrins exhibit structural diversity, flexibility and dynamism. Conformational changes, as opposed to surface expression or clustering, are central to the regulation of receptor function. In recent years, there has been intense interest in determining the three-dimensional structure of integrins, and analysing the shape changes that underpin the interconversion between functional states. Considering the central importance of the integrin signalling nexus, it is perhaps no surprise that obtaining this information has been difficult, and the answers gained so far have been complicated. In this Commentary, we pose some of the key remaining questions that surround integrin structure-function relationships and review the evidence that supports the current models.

Defining the Topology of Integrin α5β1-Fibronectin Interactions Using Inhibitory Anti-α5 and Anti-β1 Monoclonal Antibodies EVIDENCE THAT THE SYNERGY SEQUENCE OF FIBRONECTIN IS RECOGNIZED BY THE AMINO-TERMINAL REPEATS OF THE α5 SUBUNIT

The high affinity interaction of integrin α5β1 with the central cell binding domain (CCBD) of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the 10th type III repeat) and a second site (in the adjacent 9th type III repeat) which synergizes with RGD. We have attempted to map the fibronectin binding interface on α5β1 using monoclonal antibodies (mAbs) that inhibit ligand recognition. The binding of two anti-α5 mAbs (P1D6 and JBS5) to α5β1 was strongly inhibited by a tryptic CCBD fragment of fibronectin (containing both synergy sequence and RGD) but not by GRGDS peptide. Using recombinant wild type and mutated fragments of the CCBD, we show that the synergy region of the 9th type III repeat is involved in blocking the binding of P1D6 and JBS5 to α5β1. In contrast, binding of the anti-β1 mAb P4C10 to α5β1 was inhibited to a similar extent by GRGDS peptide, the tryptic CCBD fragment, or recombinant proteins lacking the synergy region, indicating that the RGD sequence is involved in blocking P4C10 binding. P1D6 inhibited the interaction of a wild type CCBD fragment with α5β1 but had no effect on the binding of a mutant fragment that lacked the synergy region. The epitopes of P1D6 and JBS5 mapped to the NH2-terminal repeats of the α5 subunit. Our results indicate that the synergy region is recognized primarily by the α5 subunit (in particular by its NH2-terminal repeats) but that the β1 subunit plays the major role in binding of the RGD sequence. These findings provide new insights into the mechanisms, specificity, and topology of integrin-ligand interactions.

Identification of a novel anti‐integrin monoclonal antibody that recognises a ligand‐induced binding site epitope on the β1 subunit

Integrins are the major family of receptors involved in the adhesive interactions of cells with extracellular matrix macromolecules. Although it is known that integrins can exist in active or inactive states, the molecular mechanisms by which integrin activity is modulated are poorly understood. A novel anti‐integrin monoclonal antibody, 12G10, that enhances α5β1‐fibronectin interactions has been identified. 12G10 binds to the β1 subunit and appears to recognise a region of the subunit that contains the epitopes of several previously described activating or inhibitory monoclonal antibodies. However, unlike other activating anti‐β1 antibodies, the binding of 12G10 to α5β1 is increased in the presence of ligands (fibronectin fragment or RGD peptide). This is the first report for the β1 integrin family of an antibody that recognises a ligand‐induced binding site, and further emphasises the functional importance of a specific region of the β1 subunit in regulating integrin‐ligand interactions.

Integrin structure: heady advances in ligand binding, but activation still makes the knees wobble.

Integrins are one of the major families of cell-adhesion receptors. In the past year, the first structure of an integrin has been published, ligand-binding pockets have been defined, and mechanisms of receptor priming and activation elucidated. Like all major advances, however, these studies have raised more questions than they have answered about issues such as the mechanisms underlying ligand-binding specificity and long-range conformational regulation.

Anti-integrin monoclonal antibodies

Integrins are a family of 24 heterodimeric transmembrane receptors that support cell-cell and cell-ECM (extracellular matrix) interactions in a multitude of physiological and disease situations ([Humphries, 2000][1]; [Hynes, 2002][2]). Adhesion that is mediated by integrins is controlled dynamically

Control of extracellular matrix assembly along tissue boundaries via Integrin and Eph/Ephrin signaling.

Extracellular matrixes (ECMs) coat and subdivide animal tissues, but it is unclear how ECM formation is restricted to tissue surfaces and specific cell interfaces. During zebrafish somite morphogenesis, segmental assembly of an ECM composed of Fibronectin (FN) depends on the FN receptor Integrinα 5β1. Using in vivo imaging and genetic mosaics, our studies suggest that incipient Itgα5 clustering along the nascent border precedes matrix formation and is independent of FN binding. Integrin clustering can be initiated by Eph/Ephrin signaling, with Ephrin reverse signaling being sufficient for clustering. Prior to activation, Itgα5 expressed on adjacent cells reciprocally and non-cell-autonomously inhibits spontaneous Integrin clustering and assembly of an ECM. Surface derepression of this inhibition provides a self-organizing mechanism for the formation and maintenance of ECM along the tissue surface. Within the tissue, interplay between Eph/Ephrin signaling, ligand-independent Integrin clustering and reciprocal Integrin inhibition restricts de novo ECM production to somite boundaries.

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.

Molecular Basis of Ligand Recognition by Integrin α5β1 I. SPECIFICITY OF LIGAND BINDING IS DETERMINED BY AMINO ACID SEQUENCES IN THE SECOND AND THIRD NH2-TERMINAL REPEATS OF THE α SUBUNIT

The NH2-terminal portion (putative ligand-binding domain) of α subunits contains 7 homologous repeats, the last 3 or 4 of which possess divalent cation binding sequences. These repeats are predicted to form a seven-bladed β-propeller structure. To map ligand recognition sites on the α5 subunit we have taken the approach of constructing and expressing αV/α5 chimeras. Although the NH2-terminal repeats of α5 and αV are >50% identical at the amino acid level, α5β1 and αVβ1show marked differences in their ligand binding specificities. Thus: (i) although both integrins recognize the Arg-Gly-Asp (RGD) sequence in fibronectin, the interaction of α5β1 but not of αVβ1 with fibronectin is strongly dependent on the “synergy” sequence Pro-His-Ser-Arg-Asn; (ii) α5β1 binds preferentially to RGD peptides in which RGD is followed by Gly-Trp (GW) whereas αVβ1 has a broader specificity; (iii) only α5β1 recognizes peptides containing the sequence Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Therefore, amino acid residues involved in ligand recognition by α5β1 can potentially be identified in gain-of-function experiments by their ability to switch the ligand binding properties of αVβ1 to those of α5β1. By introducing appropriate restriction enzyme sites, or using site-directed mutagenesis, parts of the NH2-terminal repeats of αV were replaced with the corresponding regions of the α5 subunit. Chimeric subunits were expressed on the surface of Chinese hamster ovary-B2 cells (which lack endogenous α5) as heterodimers with hamster β1. Stable cell lines were generated and tested for their ability to attach to α5β1-selective ligands. Our results demonstrate that: (a) the first three NH2-terminal repeats contain the amino acid sequences that determine ligand binding specificity and the same repeats include the epitopes of function blocking anti-α subunit mAbs; (b) the divalent cation-binding sites (in repeats 4–7) do not confer α5β1- or αVβ1-specific ligand recognition; (c) amino acid residues Ala107–Tyr226 of α5(corresponding approximately to repeats 2 and 3) are sufficient to change all the ligand binding properties of αVβ1 to those of α5β1; (d) swapping a small part of a predicted loop region of αV with the corresponding region of α5 (Asp154-Ala159) is sufficient to confer selectivity for RGDGW and the ability to recognize RRETAWA.

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.