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Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6β4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin β4 reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, suggesting a requirement for α6β4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC. Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.

Modulation of integrin function is required in many physiological and pathological settings, such as angiogenesis and cancer. Integrin allosteric changes, clustering, and trafficking cooperate to regulate cell adhesion and motility on extracellular matrix proteins via mechanisms that are partly defined. By exploiting four monoclonal antibodies recognizing distinct conformational epitopes, we show that in endothelial cells (ECs), the extracellular βI domain, but not the hybrid or I-EGF2 domain of active β1 integrins, promotes their FAK-regulated clustering into tensin 1–containing fibrillar adhesions and impairs their endocytosis. In this regard, the βI domain–dependent clustering of active β1 integrins is necessary to favor fibronectin-elicited directional EC motility, which cannot be effectively promoted by β1 integrin conformational activation alone.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6β4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin β4 using siRNA reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, indicating a requirement for α6β4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC.Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.

Retinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch’s membrane, are essential for RPE cell health and function, but the signals induced by Bruch’s membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch’s membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and β1 integrin subunits. These short-term experiments reveal an immediate protein-integrin interaction that were obtained from primary RPE cells and replicated using the hTERT-RPE1 cell line. Separate, longer term experiments utilising RNAseq analysis of hTERT-RPE1 cells bound to FHL-1, showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death suggest hTERT-RPE1 cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.

Integrin adhesion complexes (IACs) have evolved over millions of years to integrate metazoan cells physically with their microenvironment. It is presumed that the simultaneous interaction of thousands of integrin receptors to binding sites in anisotropic extracellular matrix (ECM) networks enables cells to assemble a topological description of the chemical and mechanical properties of their surroundings. This information is then converted into intracellular signals that influence cell positioning, differentiation and growth, but may also influence other fundamental processes, such as protein synthesis and energy regulation. In this way, changes in the microenvironment can influence all aspects of cell phenotype. Current concepts envisage cell fate decisions being controlled by the integrated signalling output of myriad receptor clusters, but the mechanisms are not understood. Analyses of the adhesome, the complement of proteins attracted to the vicinity of IACs, are now providing insights into some of the primordial links connecting these processes. This article reviews recent advances in our understanding of the composition of IACs, the mechanisms used to transduce signals through these junctions, and the links between IACs and cell phenotype.

Endothelial cell (EC) sensing of fluid shear stress direction is a critical determinant of vascular health and disease. Unidirectional flow induces EC alignment and vascular homeostasis, whereas bidirectional flow has pathophysiological effects. ECs express several mechanoreceptors that respond to flow, but the mechanism for sensing shear stress direction is poorly understood. We determined, by using in vitro flow systems and magnetic tweezers, that β1 integrin is a key sensor of force direction because it is activated by unidirectional, but not bidirectional, shearing forces. β1 integrin activation by unidirectional force was amplified in ECs that were pre-sheared in the same direction, indicating that alignment and β1 integrin activity has a feedforward interaction, which is a hallmark of system stability. En face staining and EC-specific genetic deletion studies in the murine aorta revealed that β1 integrin is activated and is essential for EC alignment at sites of unidirectional flow but is not activated at sites of bidirectional flow. In summary, β1 integrin sensing of unidirectional force is a key mechanism for decoding blood flow mechanics to promote vascular homeostasis. This article has an associated First Person interview with the first author of the paper., Summary: By using flow systems, magnetic tweezers and knockout mice, we show that β1 integrin is activated by unidirectional hemodynamic shear force leading to Ca2+ signalling and cell alignment, but it is not activated by bidirectional force.

Zaidel-Bar introduces work by Jones et al. demonstrating how adhesion is regulated during the cell cycle by CDK1., Adherent cells round up before division but it is unclear how detachment is regulated by the cell cycle. In this issue, Jones et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201802088) find the kinase CDK1 maintains adhesion during interphase by phosphorylating integrin adhesome proteins, including the formin FMNL2, and loss of this function of CDK1 activity in G2 triggers adhesion disassembly.

Adhesion to the extracellular matrix (ECM) persists during mitosis in most cell types. Yet, classical adhesion complexes (ACs), such as focal adhesions and focal complexes, do and must disassemble to enable cytoskeletal rearrangements associated with mitotic rounding. Given this paradox, mechanisms of mitotic cell-ECM adhesion remain undefined. Here, we identify ‘reticular adhesions’, a new class of AC that is mediated by integrin αvβ5, formed during interphase and preserved at cell-ECM attachment sites throughout cell division. Consistent with this role, integrin β5 depletion perturbs mitosis and disrupts spatial memory transmission between cell generations. Quantitative imaging reveals reticular adhesions to be both morphologically and dynamically distinct from classic focal adhesions, while mass spectrometry defines their unique composition; lacking virtually all consensus adhesome components. Indeed, remarkably, reticular adhesions are functionally independent of both talin and F-actin, yet are promoted by phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). Overall, the distinct characteristics of reticular adhesions provide a unique solution to the problem of maintaining cell-ECM attachment during mitotic rounding and division.

Adhesion to the extracellular matrix persists during mitosis in most cell types. However, while classical adhesion complexes, such as focal adhesions, do and must disassemble to enable mitotic rounding, the mechanisms of residual mitotic cell-extracellular matrix adhesion remain undefined. Here, we identify 'reticular adhesions', a class of adhesion complex that is mediated by integrin αvβ5, formed during interphase, and preserved at cell-extracellular matrix attachment sites throughout cell division. Consistent with this role, integrin β5 depletion perturbs mitosis and disrupts spatial memory transmission between cell generations. Reticular adhesions are morphologically and dynamically distinct from classical focal adhesions. Mass spectrometry defines their unique composition, enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)-binding proteins but lacking virtually all consensus adhesome components. Indeed, reticular adhesions are promoted by PtdIns(4,5)P2, and form independently of talin and F-actin. The distinct characteristics of reticular adhesions provide a solution to the problem of maintaining cell-extracellular matrix attachment during mitotic rounding and division.

The adhesion nexus is the site at which integrin receptors bridge intracellular cytoskeletal and extracellular matrix networks. The connection between integrins and the cytoskeleton is mediated by a dynamic integrin adhesion complex (IAC), the components of which transduce chemical and mechanical signals to control a multitude of cellular functions. In this Cell Science at a Glance article and the accompanying poster, we integrate the consensus adhesome, a set of 60 proteins that have been most commonly identified in isolated IAC proteomes, with the literature-curated adhesome, a theoretical network that has been assembled through scholarly analysis of proteins that localise to IACs. The resulting IAC network, which comprises four broad signalling and actin-bridging axes, provides a platform for future studies of the regulation and function of the adhesion nexus in health and disease., Summary: We have generated a new depiction of the integrin adhesome network that integrates experimentally derived IAC proteomes with the literature-curated adhesome to bridge the knowledge gap between these two resources.

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 approximately 60 A away in FN14. The IDAPS motif implicated in integrin alpha4beta1 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 alpha4beta1 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.

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking.

Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and kindlins activate β1-integrins, but the counteracting inhibiting mechanisms are poorly defined. We identified SHARPIN as an important inactivator of β1-integrins in an RNAi screen. SHARPIN inhibited β1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased β1-integrin activity, which was fully rescued by re-expression of SHARPIN. We found that SHARPIN directly binds to a conserved cytoplasmic region of integrin α-subunits and inhibits recruitment of talin and kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of β1-integrins from inactive to active conformations.

FRET experiments model integrin conformation changes in adherent cells., Integrins undergo global conformational changes that specify their activation state. Current models portray the inactive receptor in a bent conformation that upon activation converts to a fully extended form in which the integrin subunit leg regions are separated to enable ligand binding and subsequent signaling. To test the applicability of this model in adherent cells, we used a fluorescent resonance energy transfer (FRET)–based approach, in combination with engineered integrin mutants and monoclonal antibody reporters, to image integrin α5β1 conformation. We find that restricting leg separation causes the integrin to adopt a bent conformation that is unable to respond to agonists and mediate cell spreading. By measuring FRET between labeled α5β1 and the cell membrane, we find extended receptors are enriched in focal adhesions compared with adjacent regions of the plasma membrane. These results demonstrate definitely that major quaternary rearrangements of β1-integrin subunits occur in adherent cells and that conversion from a bent to extended form takes place at focal adhesions.

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. Humphries and colleagues analyse proteomic data of integrin adhesion complexes to derive a consensus integrin adhesome and characterize the temporal dynamics of adhesome component recruitment during adhesion complex assembly and disassembly.

Purpose Multipotent mesenchymal stem cells (MSCs) have the capability to differentiate down adipocyte, osteocyte and chondrocyte lineages and as such offer a range of potential therapeutic applications. The composition and stiffness of the extracellular matrix (ECM) environment that surrounds cells dictates their transcriptional programme, thereby affecting stem cell lineage decision‐making. Cells sense force via linkages between themselves and their microenvironment, and this is transmitted by integrin receptors and associated adhesion signalling complexes. To identify regulators of MSC force sensing, we sought to catalogue MSC integrin‐associated adhesion complex composition. Experimental design Adhesion complexes formed by MSCs plated on the ECM ligand fibronectin were isolated and characterised by MS. Identified proteins were interrogated by comparison to a literature‐based reference set of cell adhesion‐related components and using ontological and protein–protein interaction network analyses. Results Adhesion complex‐specific proteins in MSCs were identified that comprised predominantly cell adhesion‐related adaptors and actin cytoskeleton regulators. Furthermore, LIM domain‐containing proteins in MSC adhesion complexes were highlighted, which may act as force‐sensing components. Conclusion and clinical relevance These data provide a valuable resource of information regarding the molecular connections that link integrins and adhesion signalling in MSCs, and as such may present novel opportunities for therapeutic intervention.

The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter-receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions, and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes, together with their local plasma membrane/cytosolic environments, from cells in culture. In the first protocol, integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components via subsequent downstream analysis by western blotting or mass spectrometry.

Integrin adhesion receptors are structurally dynamic proteins that adopt a number of functionally relevant conformations. We have produced a conformation-dependent anti-alpha5 monoclonal antibody (SNAKA51) that converts alpha5beta1 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 alpha5beta1, mimicking fibrillar adhesion formation. Priming of alpha5beta1 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.

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and multiple cation-binding sites are found in both alpha and beta integrin subunits. A key cation-binding site that lies in the beta subunit A-domain is known as the metal-ion dependent adhesion site (MIDAS). Recent x-ray crystal structures of integrin alpha V beta 3 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 alpha 5 beta 1 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 beta A domain to the underlying hybrid domain, implying an important role for this site in receptor signaling.

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 alpha 5 beta 1 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 beta subunit. Fibronectin is observed to bind across the top of the head region, which contains an alpha subunit beta-propeller and a beta subunit vWF type A domain. The model of the complex indicates that the synergy region binds on the side of the beta-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 approximately 10 A.

The ligand-binding head region of integrin beta subunits contains a von Willebrand factor type A domain (betaA). Ligand binding activity is regulated through conformational changes in betaA, and ligand recognition also causes conformational changes that are transduced from this domain. The molecular basis of signal transduction to and from betaA is uncertain. The epitopes of mAbs 15/7 and HUTS-4 lie in the beta(1) subunit hybrid domain, which is connected to the lower face of betaA. Changes in the expression of these epitopes are induced by conformational changes in betaA caused by divalent cations, function perturbing mAbs, or ligand recognition. Recombinant truncated alpha(5)beta(1) with a mutation L358A in the alpha7 helix of betaA has constitutively high expression of the 15/7 and HUTS-4 epitopes, mimics the conformation of the ligand-occupied receptor, and has high constitutive ligand binding activity. The epitopes of 15/7 and HUTS-4 map to a region of the hybrid domain that lies close to an interface with the alpha subunit. Taken together, these data suggest that the transduction of conformational changes through betaA involves shape shifting in the alpha7 helix region, which is linked to a swing of the hybrid domain away from the alpha subunit.

The fibronectin (FN)-binding integrins alpha4beta1 and alpha5beta1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not. Second, adhesion via alpha5beta1 caused an eightfold increase in protein kinase Calpha (PKCalpha) activation, but only basal PKCalpha activity was observed after adhesion via alpha4beta1. Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCalpha signaling as central to the functional differences between alpha4beta1 and alpha5beta1.

The ligand-binding region of integrin beta subunits contains a von Willebrand factor type A-domain: an alpha/beta "Rossmann" fold containing a metal ion-dependent adhesion site (MIDAS) on its top face. Although there is evidence to suggest that the betaA-domain undergoes changes in tertiary structure during receptor activation, the identity of the secondary structure elements that change position is unknown. The mAb 12G10 recognizes a unique cation-regulated epitope on the beta(1) A-domain, induction of which parallels the activation state of the integrin (i.e. competency for ligand recognition). The ability of Mn(2+) and Mg(2+) to stimulate 12G10 binding is abrogated by mutation of the MIDAS motif, demonstrating that the MIDAS is a Mn(2+)/Mg(2+) binding site and that occupancy of this site induces conformational changes in the A-domain. The cation-regulated region of the 12G10 epitope maps to Arg(154)/Arg(155) in the alpha1 helix. Our results demonstrate that the alpha1 helix undergoes conformational alterations during integrin activation and suggest that Mn(2+) acts as a potent activator of beta(1) integrins because it can promote a shift in the position of this helix. The mechanism of beta subunit A-domain activation appears to be distinct from that of the A-domains found in some integrin alpha subunits.

Integrin activation, which is regulated by allosteric changes in receptor conformation, enables cellular responses to the chemical, mechanical and topological features of the extracellular microenvironment. A global view of how activation state converts the molecular composition of the region proximal to integrins into functional readouts is, however, lacking. Here, using conformation-specific monoclonal antibodies, we report the isolation of integrin activation state-dependent complexes and their characterization by mass spectrometry. Quantitative comparisons, integrating network, clustering, pathway and image analyses, define multiple functional protein modules enriched in a conformation-specific manner. Notably, active integrin complexes are specifically enriched for proteins associated with microtubule-based functions. Visualization of microtubules on micropatterned surfaces and live cell imaging demonstrate that active integrins establish an environment that stabilizes microtubules at the cell periphery. These data provide a resource for the interrogation of the global molecular connections that link integrin activation to adhesion signalling., Integrins are activated by many extracellular cues and respond by assembling diverse signalling complexes. Byron et al. use activation state-specific antibodies to proteomically characterize these complexes, and provide insight into integrin-dependent microtubule stabilization.

The tertiary structure of the integrin heterodimer is currently unknown, although several predictive models have been generated. Detailed structural studies of integrins have been consistently hampered for several reasons, including the small amounts of purified protein available, the large size and conformational flexibility of integrins, and the presence of transmembrane domains and N-linked glycosylation sites in both receptor subunits. As a first step toward obtaining crystals of an integrin receptor, we have expressed a minimized dimer. By using the Fc dimerization and mammalian cell expression system designed and optimized by Stephens et al. (Stephens, P. E., Ortlepp, S., Perkins, V. C., Robinson, M. K., and Kirby, H. (2000) Cell. Adhes. Commun. 7, 377-390), a series of recombinant soluble human alpha(5)beta(1) integrin truncations have been expressed as Fc fusion proteins. These proteins were examined for their ligand-binding properties and for their expression of anti-integrin antibody epitopes. The shortest functional alpha(5)-subunit truncation contained the N-terminal 613 residues, whereas the shortest beta(1)-subunit was a fragment containing residues 121-455. Each of these minimally truncated integrins displayed the antibody binding characteristics of alpha(5)beta(1) purified from human placenta and bound ligand with the same apparent affinity as the native receptor.

The extracellular matrix molecule fibronectin (FN) is a glycoprotein whose major functional property is to support cell adhesion. FN contains at least two distinct cell-binding domains: the central cell-binding domain and the HepII/IIICS region. The HepII region comprises type III repeats 12-14 and contains proteoglycan-binding sites, while the alternatively spliced IIICS segment possesses the major alpha4beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesion of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtained for the existence of a novel heparin-binding site in the centre of the IIICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjunction with mutations in the HepII region, caused a synergistic loss of activity. Using the H/120 variant of FN, which contains type III repeats 12-15 and the full-length IIICS region, and the H/95 variant of FN, which contains type III repeats 12-15 but lacks the high affinity integrin-binding LDV sequence, the relative roles played by cell-surface proteoglycans and integrins in mediating cell adhesion have been investigated. This was achieved by studying the effects of anti-integrin antibodies and exogenous heparin on A375 melanoma cell attachment to the wild-type and three different mutants of H/120 and H/95 in which the potential proteoglycan-binding sites were partially or completely removed. A375 cell adhesion to H/120 and its mutants was found to involve the co-operative action of both integrin and cell-surface proteoglycan binding, although integrin made a dominant contribution. Anti-integrin antibodies and exogenous heparin were capable of inhibiting melanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding.

The NH(2)-terminal portion (putative ligand-binding domain) of alpha 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 beta-propeller structure. To map ligand recognition sites on the alpha(5) subunit we have taken the approach of constructing and expressing alpha(V)/alpha(5) chimeras. Although the NH(2)-terminal repeats of alpha(5) and alpha(V) are >50% identical at the amino acid level, alpha(5)beta(1) and alpha(V)beta(1) show marked differences in their ligand binding specificities. Thus: (i) although both integrins recognize the Arg-Gly-Asp (RGD) sequence in fibronectin, the interaction of alpha(5)beta(1) but not of alpha(V)beta(1) with fibronectin is strongly dependent on the "synergy" sequence Pro-His-Ser-Arg-Asn; (ii) alpha(5)beta(1) binds preferentially to RGD peptides in which RGD is followed by Gly-Trp (GW) whereas alpha(V)beta(1) has a broader specificity; (iii) only alpha(5)beta(1) recognizes peptides containing the sequence Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Therefore, amino acid residues involved in ligand recognition by alpha(5)beta(1) can potentially be identified in gain-of-function experiments by their ability to switch the ligand binding properties of alpha(V)beta(1) to those of alpha(5)beta(1). By introducing appropriate restriction enzyme sites, or using site-directed mutagenesis, parts of the NH(2)-terminal repeats of alpha(V) were replaced with the corresponding regions of the alpha(5) subunit. Chimeric subunits were expressed on the surface of Chinese hamster ovary-B2 cells (which lack endogenous alpha(5)) as heterodimers with hamster beta(1). Stable cell lines were generated and tested for their ability to attach to alpha(5)beta(1)-selective ligands. Our results demonstrate that: (a) the first three NH(2)-terminal repeats contain the amino acid sequences that determine ligand binding specificity and the same repeats include the epitopes of function blocking anti-alpha subunit mAbs; (b) the divalent cation-binding sites (in repeats 4-7) do not confer alpha(5)beta(1)- or alpha(V)beta(1)-specific ligand recognition; (c) amino acid residues Ala(107)-Tyr(226) of alpha(5) (corresponding approximately to repeats 2 and 3) are sufficient to change all the ligand binding properties of alpha(V)beta(1) to those of alpha(5)beta(1); (d) swapping a small part of a predicted loop region of alpha(V) with the corresponding region of alpha(5) (Asp(154)-Ala(159)) is sufficient to confer selectivity for RGDGW and the ability to recognize RRETAWA.

Different beta(1) integrins bind Arg-Gly-Asp (RGD) peptides with differing specificities, suggesting a role for residues in the alpha subunit in determining ligand specificity. Integrin alpha(5)beta(1) has been shown to bind with high affinity to peptides containing an Arg-Gly-Asp-Gly-Trp (RGDGW) sequence but with relatively low affinity to other RGD peptides. The residues within the ligand-binding pocket that determine this specificity are currently unknown. A cyclic peptide containing the RGDGW sequence was found to strongly perturb the binding of the anti-alpha(5) monoclonal antibody (mAb) 16 to alpha(5)beta(1). In contrast, RGD peptides lacking the tryptophan residue acted as weak inhibitors of mAb 16 binding. The epitope of mAb 16 has previously been localized to a region of the alpha(5) subunit that contains Ser(156)-Trp(157). Mutation of Trp(157) (but not of Ser(156) or surrounding residues) to alanine blocked recognition of mAb 16 and perturbed the high affinity binding of RGDGW-containing peptides to alpha(5)beta(1). The same mutation also abrogated recognition of the alpha(5)beta(1)-specific ligand peptide Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Based on these findings, we propose that Trp(157) of alpha(5) participates in a hydrophobic interaction with the tryptophan residue in RGDGW, and that this interaction determines the specificity of alpha(5)beta(1) for RGDGW-containing peptides. Since the RGD sequence is recognized predominantly by amino acid residues on the beta(1) subunit, our results suggest that Trp(157) of alpha(5) must lie very close to these residues. Our findings therefore provide new insights into the structure of the ligand-binding pocket of alpha(5)beta(1).

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

In this study, we identified an adhesion-regulated subunit of the interleukin-1 (IL-1) receptor complex. Transfection of fibroblasts with an IL-1 receptor-EGFP construct showed that the fusion protein was located at focal adhesions in cells attaching to fibronectin. Fibronectin attachment caused enhancement in endogenous IL-1 type I receptor levels from on average 2500 to 4300 receptors/cell. In addition, matrix attachment resulted in a decrease in binding affinity (Ka) from 1.0 x 10(9) (M-1) to 5.6 x 10(8) (M-1), due to a 2-fold reduction in association rate constant. The adhesion-mediated effects were reversed by soluble heparin. Cross-linking experiments showed that in cells attached to fibronectin, 50-70% of the radiolabeled IL-1 was associated with a heparinase sensitive, high molecular mass component of about 300 kDa, with a core protein of 80-90 kDa. Formation of the complex was dependent on cell interaction with the heparin binding region in fibronectin and required IL-1/type I IL-1 receptor binding. This report demonstrates the recruitment of a heparan sulfate to the IL-1 receptor complex, following attachment to fibronectin, which correlates with alterations in receptor function. The data suggest that the heparan sulfate constitutes an attachment regulated component of the IL-1 receptor complex with the role of mediating matrix regulation of IL-1 responses.

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.

The extracellular matrix (ECM) is a network of macromolecules that underlies all epithelia and endothelia and that surrounds all connective tissue cells. The ECM provides mechanical support and also profoundly influences the behavior and the differentiation state of cells in contact with it. The main receptors mediating the interaction of cells with ECM proteins are known as integrins, referring to their function of integrating the cell's exterior with its interior. Following ligand binding, the cytoplasmic domains of integrins connect to the cytoskeleton and trigger the assembly of signaling complexes. Conversely, the binding of intracellular cytoplasmic components influences cell adhesiveness by altering integrin conformation. Thus, a large variety of complex signaling events can be transduced by integrins in a bidirectional manner across the cell membrane. These events serve to modulate and coordinate many aspects of cell behavior, such as proliferation, survival, shape, polarity, motility, gene expression, and differentiation that are required for fundamental processes such as development, tissue morphogenesis, and wound healing within multicellular organisms. Integrins are also implicated in several disease processes such as inflammation, thrombosis, and cancer metastasis, whereas mutations in integrin genes lead to deficiencies in leukocyte adhesion, myopathy, and blistering skin diseases.

Activation of hepatic stellate cells (HSCs) and subsequent uncontrolled accumulation of altered extracellular matrix (ECM) underpin liver fibrosis, a wound healing response to chronic injury, which can lead to organ failure and death. We sought to catalogue the components of fibrotic liver ECM to obtain insights into disease etiology and aid identification of new biomarkers. Cell-derived ECM was isolated from the HSC line LX-2, an in vitro model of liver fibrosis, and compared to ECM from human foreskin fibroblasts (HFFs) as a control. Mass spectrometry analyses of cell-derived ECMs identified, with ≥99% confidence, 61 structural ECM or secreted proteins (48 and 31 proteins for LX-2 and HFF, respectively). Gene ontology enrichment analysis confirmed the enrichment of ECM proteins, and hierarchical clustering coupled with protein-protein interaction network analysis revealed a subset of proteins enriched to fibrotic ECM, highlighting the existence of cell type-specific ECM niches. Thirty-six proteins were enriched to LX-2 ECM as compared to HFF ECM, of which Wnt-5a and CYR61 were validated by immunohistochemistry in human and murine fibrotic liver tissue. Future studies will determine if these and other components may play a role in the etiology of hepatic fibrosis, serve as novel disease biomarkers, or open up new avenues for drug discovery.

The integrin receptor alpha 4 beta 1 (also known as VLA-4) binds two different ligands, the endothelial cell surface protein vascular cell adhesion molecule-1 (VCAM-1) and the extracellular matrix component fibronectin. Three distinct sites in fibronectin are recognized by alpha 4 beta 1. Two of these (represented by peptides CS1 and CS5) are present in the alternatively spliced IIICS region and lie in separate, independently spliced segments of this region. A third site resides in the adjacent constitutively expressed HepII domain. Recombinant proteins containing the HepII domain and different splice variants of the IIICS have been generated and compared for their ability to mediate cell attachment, spreading and migration. The activity of these proteins has also been compared with that of a recombinant soluble form of VCAM-1 (rsVCAM-1). All the recombinant proteins supported A375-SM human melanoma cell attachment and spreading in an alpha 4 beta 1-dependent manner, but had varied adhesive activities with rsVCAM-1 > fibronectin variants containing the CS1 sequence >> other fibronectin variants. Low concentrations of rsVCAM-1 and CS1-containing fibronectin variants effectively supported cell migration in a trans-filter assay; however, cell motility was retarded at high concentrations of the same proteins. Fibronectin variants lacking CS1 supported little or no migration. To obtain further insight into the molecular basis of this varied adhesive activity, apparent dissociation constants for each of the recombinant proteins were measured using a solid phase receptor-ligand binding assay. The results revealed a hierarchy of ligand affinities that mirrored their adhesive activity (rsVCAM-1 > fibronectin variants containing CS1 >> other fibronectin variants).

The integrin receptor alpha 4 beta 1 binds to two different ligands, the extracellular matrix glycoprotein fibronectin and the endothelial cell surface protein vascular cell adhesion molecule-1 (VCAM-1). Using probes derived from each ligand and a variety of cell adhesion and ligand-receptor binding assays, we have investigated the relationship between the mechanisms of fibronectin and VCAM-1 interaction with alpha 4 beta 1. CS1 peptide, which represents the dominant active site from the HepII/IIICS recognition domain in fibronectin, was found to inhibit VCAM-1-dependent adhesion in three different assays: MOLT-4 T lymphoblastic leukaemia cell attachment to immobilized recombinant soluble VCAM-1 (rsVCAM-1), MOLT-4 cell attachment to monolayers of VCAM-1-transfected COS-1 cells, and A375-SM melanoma cell spreading on immobilized rs VCAM-1. Half-maximal inhibition required CS1 concentrations of 1.7-3.0 mg/ml, some 3-7-fold higher than that needed to autoinhibit adhesion to CS1-IgG conjugate. Using a more sensitive solid-phase receptor-ligand binding assay, CS1 was found to be a potent inhibitor of the binding of rsVCAM-1 to alpha 4 beta 1 (half-maximal inhibition at 13 micrograms/ml). In agreement with cell-based assays, severalfold lower concentrations of CS1 were required to inhibit binding of recombinant HepII/IIICS region of fibronectin (half-maximal inhibition at 3 micrograms/ml). VCAM-1-alpha 4 beta 1 binding was blocked not only by CS1 peptide but also by the recombinant HepII/IIICS region of fibronectin. Kinetic analysis of CS1 inhibition of VCAM-1 binding revealed that it was directly competitive in nature, indicating that VCAM-1 and fibronectin recognize either identical or spatially overlapping binding sites on alpha 4 beta 1. The implications of these results for the future design of VCAM-1 antagonists are discussed.

Integrins are divalent cation-dependent, αβ heterodimeric adhesion receptors that control many fundamental aspects of cell behaviour by bi-directional signalling between the extracellular matrix and intracellular cytoskeleton. The activation state of cell surface integrins is tightly regulated by divalent cation occupancy of the ligand-binding pocket and by interaction with cytoplasmic adaptor proteins, such as talin. These agents elicit gross conformational changes across the entire molecule, which specify the activation state. Much less is known about the activation state of newly synthesised integrins or the role of cations during the early folding and trafficking of integrins. Here we use a number of well-characterised, conformation-specific antibodies to demonstrate that β1-integrins adopt the bent, inactive conformation after assembly with α-integrins in the endoplasmic reticulum. Folding and assembly are totally dependent on the binding of Ca2+ ions. In addition, Ca2+ binding prevents integrin activation before its arrival at the cell surface. Activation at the cell surface occurs only following displacement of Ca2+ with Mg2+ or Mn2+. These results demonstrate the essential roles played by divalent cations to facilitate folding of the β-integrin subunit, to prevent inappropriate intracellular integrin signalling, and to activate ligand binding and signalling at the cell surface.

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.

Fibronectin (FN) is a prototypic adhesive glycoprotein that is widely expressed in extracellular matrices and body fluids. The fibronectin molecule is dimeric, and composed of a series of repeating polypeptide modules. A recombinant fragment of FN incorporating type III repeats 12-15, and including the alternatively-spliced type three connecting segment (IIICS), was found to bind Ni(2+), Cu(2+) and Zn(2+) divalent cations, whereas a similar fragment lacking the IIICS did not. Mutation of two pairs of histidine residues in separate spliced regions of the IIICS reduced cation binding to near the level of the variant lacking the IIICS, suggesting a zinc finger-like mode of cation coordination. Analysis of native FNs purified from plasma or amniotic fluid revealed significant levels of zinc associated with those isoforms that contain the complete IIICS. Taken together, these data demonstrate that the IIICS region of FN is a novel zinc-binding module.

The overall structure of integrins is that of a ligand-binding head connected to two long legs. The legs can exhibit a pronounced bend at the "knees," and it has been proposed that the legs undergo a dramatic straightening when integrins transit from a low affinity to a high affinity state. The knee region contains domains from both alpha and beta subunits, including the N-terminal plexin/semaphorin/integrin (PSI) domain of the beta subunit. The role played by the knee domains in the regulation of integrin-ligand binding is uncertain. Here we show that: (i) monoclonal antibodies (mAbs) N29 and 8E3 have epitopes in the beta(1) subunit PSI domain and stimulate ligand binding to alpha(5)beta(1); (ii) N29 and 8E3 cause long range conformational changes that alter the ligand binding activity of the head region; (iii) the stimulatory action of these mAbs is dependent on the calf-1 domain, which forms part of the alpha subunit knee; and (iv) the epitopes of 8E3 and N29 map close to the extreme N terminus of the PSI and are likely to lie on the side of this domain that faces the alpha subunit. Taken together, our data suggest that the binding of these mAbs results in a levering apart of the PSI and calf-1 domains, and thereby causes the alpha and beta subunit knees to separate. Several major inferences can be drawn from our findings. First, the PSI domain appears to form part of an interface with the alpha subunit that normally restrains the integrin in a bent state. Second, the PSI domain is important for the transduction of conformational changes from the knee to head. Third, unbending is likely to provide a general mechanism for control of integrin-ligand recognition.

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.

The interaction of fibronectin with cell surface heparan sulfate proteoglycans is important biologically in inducing reorganization of the cytoskeleton and the assembly of focal adhesions. The major heparan sulfate- binding site in fibronectin, which is also implicated in these morphological events, is the COOH-terminal Hep-2 domain. We describe the first extensive study of the structural determinants required for the interaction between heparan sulfate/heparin and Hep-2. It is clear that, in heparan sulfate, there is a very prominent role for N-sulfate groups, as opposed to a relatively small apparent contribution from carboxyl groups. Furthermore, a minimal octasaccharide binding sequence appeared to contain at least two 2-O- sulfated iduronate residues, but no 6-O-sulfate groups. However, affinity was enhanced by the presence of 6-O-sulfates, and the interaction with Hep-2 also increased progressively with oligosaccharide size up to a maximum length of a tetradecasaccharide. This overall specificity is compatible with recent information on the structure of Hep-2 (Sharma, A., Askari, J. A., Humphries, M. J., Jones, E. Y., and Stuart, D. I. (1999) EMBO J. 18, 1468-1479) in which two separate, positively charged clusters, involving up to 11 basic amino acid residues (mostly arginines with their preferential ability to co- ordinate sulfate groups), could form a single extended binding site.

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 alpha 5 beta 1-fibronectin interactions has been identified. 12G10 binds to the beta 1 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-beta 1 antibodies, the binding of 12G10 to alpha 5 beta 1 is increased in the presence of ligands (fibronectin fragment or RGD peptide). This is the first report for the beta 1 integrin family of an antibody that recognises a ligand-induced binding site, and further emphasises the functional importance of a specific region of the beta 1 subunit in regulating integrin-ligand interactions.

Integrin alpha2beta1 is the major receptor for collagens in the human body, and the collagen-binding site on the alpha2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant alpha2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse alpha2beta1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr(221) to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-beta1 antibody Mab13, and binding of Gi9 and JA218 to alpha2beta1 was inhibited by substituting Mn(2+) for Mg(2+), demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212-216, while JA208 bound between residues 199-216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.

Experimental in vitro models that capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel extracellular matrix designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from genetically engineered murine models and human patients, essential adhesive cues were empirically defined and replicated in the hydrogel scaffold, revealing a functional role of laminin–integrin α3/α6 signalling in establishment and survival of pancreatic organoids. Altered tissue stiffness—a hallmark of pancreatic cancer—was recapitulated in culture by adjusting the hydrogel properties to engage mechano-sensing pathways and alter organoid growth. Pancreatic stromal cells were readily incorporated into the hydrogels and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro. A synthetic hydrogel has been developed to mimic the physicochemical properties of pancreatic tissue and is shown to support the culture of pancreatic cancer organoids, revealing the role of laminin–integrin interactions in their growth.