Janet A. Askari

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.

Crystal structure of a heparin- and integrin-binding segment of human fibronectin.

The crystal structure of human fibronectin (FN) type III repeats 12–14 reveals the primary heparin‐binding site, a clump of positively charged residues in FN13, and a putative minor site ∼60 Å away in FN14. The IDAPS motif implicated in integrin α4β1 binding is at the FN13–14 junction, rendering the critical Asp184 inaccessible to integrin. Asp184 clamps the BC loop of FN14, whose sequence (PRARI) is reminiscent of the synergy sequence (PHSRN) of FN9. Mutagenesis studies prompted by this observation reveal that both arginines of the PRARI sequence are important for α4β1 binding to FN12–14. The PRARI motif may represent a new class of integrin‐binding sites. The spatial organization of the binding sites suggests that heparin and integrin may bind in concert.

Integrin-specific signaling pathways controlling focal adhesion formation and cell migration

The fibronectin (FN)-binding integrins α4β1 and α5β1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of α4+/α5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with α4β1 and α5β1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, α5β1 and α4β1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; α5β1 requires a proteoglycan coreceptor (syndecan-4), and α4β1 does not. Second, adhesion via α5β1 caused an eightfold increase in protein kinase Cα (PKCα) activation, but only basal PKCα activity was observed after adhesion via α4β1. Pharmacological inhibition of PKCα and transient expression of dominant-negative PKCα, but not dominant-negative PKCδ or PKCζ constructs, suppressed focal adhesion formation and cell migration mediated by α5β1, but had no effect on α4β1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCα signaling as central to the functional differences between α4β1 and α5β1.

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.

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

Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly

Integrin receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this data set reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome is likely to represent a core cell adhesion machinery, centred around four axes comprising ILK–PINCH–kindlin, FAK–paxillin, talin–vinculin and α-actinin–zyxin–VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community.

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.