A P Mould

Regulation of integrin alpha 5 beta 1-fibronectin interactions by divalent cations. Evidence for distinct classes of binding sites for Mn2+, Mg2+, and Ca2+

Integrin-ligand interactions are known to be dependent on divalent cations, although the precise role of cations in ligand binding is still unclear. Using the interaction between α5β1 and fibronectin as a model system, we have performed a comprehensive analysis of the effects of Mn2+, Mg2+, and Ca2+ on ligand binding. Each cation had distinct effects on the ligand-binding capacity of α5β1: Mn2+ promoted high levels of ligand binding, Mg2+ promoted low levels of binding, and Ca2+ failed to support binding. Studies of the effects of different combinations of cations on ligand binding indicated that the cation-binding sites within α5β1 are not all identical, or of broad specificity, but instead each site shows a distinct preference for one or more cations. Ca2+ strongly inhibited Mn2+-supported ligand binding, but this inhibition was noncompetitive, suggesting that Ca2+ recognizes different cation-binding sites to Mn2+. In contrast, Ca2+ acted as a direct competitive inhibitor of Mg2+-supported ligand binding, implying that Ca2+ can displace Mg2+ from the integrin. However, low concentrations of Ca2+ greatly increased the apparent affinity of Mg2+ for its binding site, suggesting the existence of a distinct high affinity Ca2+-binding site. Taken together, our results imply that the ligand-binding capacity of α5β1 can be regulated in a complex manner through separate classes of binding sites for Mn2+, Mg2+, and Ca2+.

The cell-binding domain of intimin from enteropathogenic Escherichia coli binds to beta1 integrins

Bacteria interact with mammalian cells surface molecules, such as integrins, to colonize tissues and evade immunological detection. Herein, the ability of intimin, an outer membrane protein from enteropathogenic Escherichia coli, to bind β1 integrins was investigated. Solid-phase binding assays revealed binding of the carboxyl-terminal 280 amino acids of intimin (Int280) to α4β1 and α5β1 integrins. The binding required divalent ions (in particular, it was enhanced by Mn2+) and was inhibited by an RGD-containing peptide. Nonderivatized Int280, but not Int280CS (like Int280 but with Cys-937 replaced by Ser) blocked the binding of biotinylated Int280 to integrins. Int280 did not efficiently inhibit β1 integrin binding of invasin from Yersinia pseudotuberculosis. Both intimin and invasin, immobilized on plastic surfaces, mediated adherence of resting or phorbol 12-myristate 13-acetate-activated human CD4+ T cells, whereas fibronectin mediated the adherence of only activated T cells. T cell binding to intimin and invasin was integrin mediated because it was specifically blocked by an RGD-containing peptide and by antibodies directed against the integrin subunits β1, α4, and α5. These results demonstrate a specific integrin binding activity for intimin that is related to, but distinct from, that of invasin.

Identification of a novel recognition sequence for the integrin alpha 4 beta 1 in the COOH-terminal heparin-binding domain of fibronectin

The type III connecting segment of fibronectin contains two cell binding sites, represented by the peptides CS1 and CS5, that are recognized by the integrin receptor alpha 4 beta 1. Using assays measuring the spreading of A375‐SM human melanoma cells, we now report that the adhesion promoting activity of a 29 kDa protease fragment of fibronectin containing the COOH‐terminal heparin‐binding domain (HepII), but lacking CS1 and CS5, is completely sensitive to anti‐alpha 4 and anti‐beta 1 antibodies, suggesting that HepII contains a third alpha 4 beta 1‐binding sequence. Examination of the primary structure of HepII revealed a sequence with homology to CS1. A 19mer peptide spanning this region (designated H1) was found to support cell spreading to the same level as the 29 kDa fragment. H1‐dependent adhesion was completely sensitive to anti‐alpha 4 and anti‐beta 1 antibodies. When soluble peptides were tested for their ability to block cell spreading on the 29 kDa fragment, a 13mer peptide comprising the central core of H1 was found to be completely inhibitory. The active region of H1 was localized to the pentapeptide IDAPS, which is homologous to LDVPS from the active site of CS1. Taken together, these results identify a novel peptide sequence in the HepII region of fibronectin that supports alpha 4 beta 1‐dependent cell adhesion.

The inhibitory anti-beta1 integrin monoclonal antibody 13 recognizes an epitope that is attenuated by ligand occupancy. Evidence for allosteric inhibition of integrin function

Integrin-ligand binding causes conformational changes in the integrin, as evidenced by the increased expression of epitopes known as ligand-induced binding sites. Some monoclonal antibodies (mAbs) that recognize ligand-induced binding sites stimulate ligand binding, possibly by stabilizing the ligand-occupied conformation of the integrin. Here we have investigated the effect of ligand recognition by α5β1 on the binding of a mAb that inhibits β1 integrin function (mAb 13). Ligand (fibronectin fragment or GRGDS peptide) decreased the binding of mAb 13 to α5β1. Analysis of this inhibition showed that at high ligand concentrations, approximately 50% of the total integrin bound mAb 13 with >50-fold lower affinity than in the absence of ligand. The concentration of ligand required for half-maximal inhibition of antibody binding was independent of antibody concentration, suggesting that ligand acts as an allosteric inhibitor of mAb 13 binding. Hence, ligand and mAb 13 did not appear to compete directly for binding to α5β1. The stimulatory anti-β1 mAb 9EG7 was found to increase the maximum level of ligand binding ∼2-fold, indicating that up to 50% of the total integrin could not bind ligand without 9EG7 stimulation. Analysis of mAb 13 binding in the presence of 9EG7 and ligand (i.e. maximal ligand occupancy) demonstrated that essentially all of the integrin bound mAb 13 with very low or zero affinity. Our results demonstrate that mAb 13 recognizes an epitope that is dramatically attenuated in the ligand-occupied form of α5β1. Hence, since mAb 13 preferentially recognizes the unoccupied conformation of the integrin, the antibody may inhibit ligand binding by stabilizing the unoccupied state of α5β1. In addition, we present evidence that the binding of mAb 13 to ligand-occupied α5β1 may also induce a conformational change in the integrin, resulting in the displacement of ligand.

Mechanisms of integration of cells and extracellular matrices by integrins.

While it is self-evident that all extracellular molecules are an integral part of a multicellular organism, it is paradoxical that they are often considered to be dissociated from cells. The reality is that a continuum of dynamic, bi-directional interactions links the intracellular environment through cell-surface receptors to multimolecular extracellular assemblies. These interactions not only control the behaviour of individual cells, but also determine tissue architecture. Adhesion receptor function is partly determined by an ability to tether the contractile cytoskeleton to the plasma membrane, but there is also evidence that integrin receptors modulate signalling events that are essential for cellular differentiation. A major challenge is now to integrate work at the atomic, molecular and cellular levels, and obtain holistic insights into the mechanisms controlling cell adhesion. In the present study, we review current knowledge of the molecular mechanisms employed by cells to integrate with the extracellular matrix. Two main topics are covered: the adaptation of integrin structure for bi-directional signalling and the integration of integrin signalling with other receptors.

Foot-and-mouth disease virus is a ligand for the high-affinity binding conformation of integrin alpha5beta1: influence of the leucine residue within the RGDL motif on selectivity of integrin binding

Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors for internalization, whereas strains that are adapted for growth in cultured cell lines appear to be able to use alternative receptors like heparan sulphate proteoglycans (HSPG). The ligand-binding potential of integrins is regulated by changes in the conformation of their ectodomains and the ligand-binding state would be expected to be an important determinant of tropism for viruses that use integrins as cellular receptors. Currently, alphavbeta3 is the only integrin that has been shown to act as a receptor for FMDV. In this study, a solid-phase receptor-binding assay has been used to characterize the binding of FMDV to purified preparations of the human integrin alpha5beta1, in the absence of HSPG and other RGD-binding integrins. In this assay, binding of FMDV resembled authentic ligand binding to alpha5beta1 in its dependence on divalent cations and specific inhibition by RGD peptides. Most importantly, binding was found to be critically dependent on the conformation of the integrin, as virus bound only after induction of the high-affinity ligand-binding state. In addition, the identity of the amino acid residue immediately following the RGD motif is shown to influence differentially the ability of FMDV to bind integrins alpha5beta1 and alphavbeta3 and evidence is provided that alpha5beta1 might be an important FMDV receptor in vivo.

THE INTEGRIN BETA SUBUNIT

The integrin family of cell adhesion receptors plays a fundamental role in the processes involved in cell division, differentiation and movement. The extracellular domains of integrin alpha/beta heterodimers mediate cell-matrix and cell-cell contacts while their cytoplasmic tails associate with the cytoskeleton. Integrins are capable of transducing information in a bidirectional manner and the beta subunit is now recognised to play an important role in this process. Recent studies have led to the identification of a ligand-binding region on the beta subunit similar to that already characterised on some alpha subunits, and sequences in the cytoplasmic tails of the beta subunits that interact with cytoskeletal and signalling components. Adhesive events can also play a role in the progression of all four major classes of human disease--neoplastic, inflammatory, traumatic and infectious--and the specific nature of integrin adhesion mechanisms make them an attractive target for therapy.

Identification of amino acid residues that form part of the ligand-binding pocket of integrin alpha5 beta1

Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel ligand peptide for integrin α5β1, which blocks α5β1-mediated cell adhesion to fibronectin (Koivunen, E., Wang, B., and Ruoslahti, E. (1994)J. Cell Biol. 124, 373–380). Here we have localized the binding site for RRETAWA on α5β1 using inhibitory monoclonal antibodies (mAbs) and site-directed mutagenesis. A cyclic peptide containing this sequence (*CRRETAWAC*) had little effect on the binding of most anti-α5 and anti-β1 mAbs to α5β1 but completely blocked binding of the anti-α5 mAb 16 in a directly competitive manner. Hence, the binding site of RRETAWA appears to closely overlap with the epitope of mAb 16. *CRRETAWAC* also acted as a direct competitive inhibitor of the binding of Arg-Gly-Asp (RGD)-containing fibronectin fragments to α5β1, suggesting that the binding site for RRETAWA is also closely overlapping with that for RGD. However, differences between the binding sites of RRETAWA and RGD were apparent in that (i) RGD peptides allosterically inhibited the binding of mAb 16 to α5β1, and (ii) several mAbs that perturbed binding of α5β1 to RGD had little effect on binding of α5β1 to RRETAWA. A double mutation in α5 (S156G/W157S) blocked the interaction of both RRETAWA and mAb 16 with α5β1 but had no effect on fibronectin binding or on the binding of other anti-α5 mAbs. Ser156-Trp157 is located near the apex of a putative loop region on the upper surface of a predicted β-propeller structure formed by the NH2-terminal repeats of α5. Our findings suggest that this sequence forms part of the ligand-binding pocket of α5β1. Furthermore, as Ser156-Trp157 is unique to the α5 subunit, it may be responsible for the specific recognition of RRETAWA by α5β1.