Interactions of Soluble Recombinant Integrin αvβ5 with Human Adenoviruses

αv integrins have been identified as coreceptors for adenovirus (Ad) internalization; however, direct interactions of these molecules with Ad have not been demonstrated. We report here the expression of soluble integrin αvβ5, which retains the ability to recognize the Ad penton base as well as vitronectin, an Arg Gly Asp (RGD)-containing extracellular matrix protein. Soluble integrin αvβ5 reacted with seven different Ad serotypes (subgroups A to E) in solid-phase binding assays. The soluble integrin exhibited different levels of binding to each Ad serotype; however, binding to multiple Ad types required the presence of divalent metal cations and was inhibited by a synthetic RGD peptide, indicating that RGD and cation-binding sequences regulate Ad interactions with αvβ5. Incubation of Ad particles with soluble αvβ5 integrin also inhibited subsequent Ad internalization into epithelial cells as well as virus attachment to monocytic cells. These findings suggest that soluble αv integrins or antagonists of these coreceptors could be used to limit infection by multiple Ad types. The generation of soluble αv integrins should also permit further detailed kinetic and structural analysis of Ad interactions with its coreceptors.     

Foot-and-mouth disease virus forms a highly stable, EDTA-resistant complex with its principal receptor, integrin alphavbeta6: implications for infectiousness

The initial stage of foot-and-mouth disease virus (FMDV) infection is virus binding to cell surface integrins via the RGD motif in the GH loop of the VP1 capsid protein. As for all ligand/integrin interactions, the initial contact between FMDV and its integrin receptors is cation dependent and hence inhibited by EDTA. We have investigated this binding process with RGD-containing peptides derived from the VP1 capsid protein of FMDV and discovered that, upon binding, some of these peptides form highly stable, EDTA-resistant associations with integrin αvβ6. Peptides containing specific substitutions show that this stable binding is dependent on a helical structure immediately C terminal to the RGD and, specifically, two leucine residues at positions RGD +1 and RGD +4. These observations have a biological consequence, as we show further that stable, EDTA-resistant binding to αvβ6 is a property also exhibited by FMDV particles. Thus, the integrin-binding loop of FMDV appears to have evolved to form very stable complexes with the principal receptor of FMDV, integrin αvβ6. An ability to induce such stable complexes with its cellular receptor is likely to contribute significantly to the high infectiousness of FMDV.     

Cell surface-expressed phosphatidylserine as therapeutic target to enhance phagocytosis of apoptotic cells

Impaired efferocytosis has been shown to be associated with, and even to contribute to progression of, chronic inflammatory diseases such as atherosclerosis. Enhancing efferocytosis has been proposed as strategy to treat diseases involving inflammation. Here we present the strategy to increase ‘eat me'' signals on the surface of apoptotic cells by targeting cell surface-expressed phosphatidylserine (PS) with a variant of annexin A5 (Arg-Gly-Asp–annexin A5, RGD–anxA5) that has gained the function to interact with α_vβ₃ receptors of the phagocyte. We describe design and characterization of RGD–anxA5 and show that introduction of RGD transforms anxA5 from an inhibitor into a stimulator of efferocytosis. RGD–anxA5 enhances engulfment of apoptotic cells by phorbol-12-myristate-13-acetate-stimulated THP-1 (human acute monocytic leukemia cell line) cells in vitro and resident peritoneal mouse macrophages in vivo. In addition, RGD–anxA5 augments secretion of interleukin-10 during efferocytosis in vivo, thereby possibly adding to an anti-inflammatory environment. We conclude that targeting cell surface-expressed PS is an attractive strategy for treatment of inflammatory diseases and that the rationally designed RGD–anxA5 is a promising therapeutic agent.     

Interactions between Growth Factors and Integrins: Latent Forms of Transforming Growth Factor-β Are Ligands for the Integrin αvβ1

The multipotential cytokine transforming growth factor-β (TGF-β) is secreted in a latent form. Latency results from the noncovalent association of TGF-β with its processed propeptide dimer, called the latency-associated peptide (LAP); the complex of the two proteins is termed the small latent complex. Disulfide bonding between LAP and latent TGF-β–binding protein (LTBP) produces the most common form of latent TGF-β, the large latent complex. The extracellular matrix (ECM) modulates the activity of TGF-β. LTBP and the LAP propeptides of TGF-β (isoforms 1 and 3), like many ECM proteins, contain the common integrin-binding sequence RGD. To increase our understanding of latent TGF-β function in the ECM, we determined whether latent TGF-β1 interacts with integrins. A549 cells adhered and spread on plastic coated with LAP, small latent complex, and large latent complex but not on LTBP-coated plastic. Adhesion was blocked by an RGD peptide, and cells were unable to attach to a mutant form of recombinant LAP lacking the RGD sequence. Adhesion was also blocked by mAbs to integrin subunits αv and β1. We purified LAP-binding integrins from extracts of A549 cells using LAP bound to Sepharose. αvβ1 eluted with EDTA. After purification in the presence of Mn²⁺, a small amount of αvβ5 was also detected. A549 cells migrated equally on fibronectin- and LAP-coated surfaces; migration on LAP was αvβ1 dependent. These results establish αvβ1 as a LAP-β1 receptor. Interactions between latent TGF-β and αvβ1 may localize latent TGF-β to the surface of specific cells and may allow the TGF-β1 gene product to initiate signals by both TGF-β receptor and integrin pathways.     

Integrin α3β1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat

Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbβ3 and α5β1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3β1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu → Pro) stabilizing mutant (FnIII9′10), (ii) an Arg to Ala synergy site mutation (FnIII9^R→^A10), (iii) a two-Gly (FnIII9^2G10) insertion, and (iv) a four-Gly (FNIII9^4G10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3β1 to the Fn fragments. Integrin α3β1 had the highest affinity for FnIII9′10 and FnIII9^2G10. Mutation within the synergy site decreased integrin α3β1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9′10. Cell attachment studies demonstrate that α3β1-mediated epithelial cell binding is greater on FnIII9′10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3β1 binding to Fn.     

Induction of the Angiogenic Phenotype by Hox D3

Angiogenesis is characterized by distinct phenotypic changes in vascular endothelial cells (EC). Evidence is provided that the Hox D3 homeobox gene mediates conversion of endothelium from the resting to the angiogenic/invasive state. Stimulation of EC with basic fibroblast growth factor (bFGF) resulted in increased expression of Hox D3, integrin αvβ3, and the urokinase plasminogen activator (uPA). Hox D3 antisense blocked the ability of bFGF to induce uPA and integrin αvβ3 expression, yet had no effect on EC cell proliferation or bFGF-mediated cyclin D1 expression. Expression of Hox D3, in the absence of bFGF, resulted in enhanced expression of integrin αvβ3 and uPA. In fact, sustained expression of Hox D3 in vivo on the chick chorioallantoic membrane retained EC in this invasive state and prevented vessel maturation leading to vascular malformations and endotheliomas. Therefore, Hox D3 regulates EC gene expression associated with the invasive stage of angiogenesis.     

Integrin-mediated Cell Attachment Induces a PAK4-dependent Feedback Loop Regulating Cell Adhesion through Modified Integrin αvβ5 Clustering and Turnover

Cell-to-extracellular matrix adhesion is regulated by a multitude of pathways initiated distally to the core cell–matrix adhesion machinery, such as via growth factor signaling. In contrast to these extrinsically sourced pathways, we now identify a regulatory pathway that is intrinsic to the core adhesion machinery, providing an internal regulatory feedback loop to fine tune adhesion levels. This autoinhibitory negative feedback loop is initiated by cell adhesion to vitronectin, leading to PAK4 activation, which in turn limits total cell–vitronectin adhesion strength. Specifically, we show that PAK4 is activated by cell attachment to vitronectin as mediated by PAK4 binding partner integrin αvβ5, and that active PAK4 induces accelerated integrin αvβ5 turnover within adhesion complexes. Accelerated integrin turnover is associated with additional PAK4-mediated effects, including inhibited integrin αvβ5 clustering, reduced integrin to F-actin connectivity and perturbed adhesion complex maturation. These specific outcomes are ultimately associated with reduced cell adhesion strength and increased cell motility. We thus demonstrate a novel mechanism deployed by cells to tune cell adhesion levels through the autoinhibitory regulation of integrin adhesion.     

αvβ3 Integrin Boosts the Innate Immune Response Elicited in Epithelial Cells through Plasma Membrane and Endosomal Toll-Like Receptors

We report that αvβ3 integrin strongly affects the innate immune response in epithelial cells. αvβ3 integrin greatly increased the response elicited via plasma membrane Toll-like receptors (TLRs) by herpes simplex virus or bacterial ligands. The endosomal TLR3, not the cytosolic sensor interferon gamma-inducible protein 16 (IFI16), was also boosted by αvβ3 integrin. The boosting was exerted specifically by αvβ3 integrin but not by αvβ6 or αvβ8 integrin. Current and previous work indicates that integrin-TLR cooperation occurs in epithelial and monocytic cells. The TLR response should be considered an integrin-TLR response.     

Identification of an Endogenously Generated Cryptic Collagen Epitope (XL313) That May Selectively Regulate Angiogenesis by an Integrin Yes-associated Protein (YAP) Mechano-transduction Pathway

Extracellular matrix (ECM) remodeling regulates angiogenesis. However, the precise mechanisms by which structural changes in ECM proteins contribute to angiogenesis are not fully understood. Integrins are molecules with the ability to detect compositional and structural changes within the ECM and integrate this information into a network of signaling circuits that coordinate context-dependent cell behavior. The role of integrin αvβ3 in angiogenesis is complex, as evidence exists for both positive and negative functions. The precise downstream signaling events initiated by αvβ3 may depend on the molecular characteristics of its ligands. Here, we identified an RGD-containing cryptic collagen epitope that is generated in vivo. Surprisingly, rather than inhibiting αvβ3 signaling, this collagen epitope promoted αvβ3 activation and stimulated angiogenesis and inflammation. An antibody directed to this RGDKGE epitope but not other RGD collagen epitopes inhibited angiogenesis and inflammation in vivo. The selective ability of this RGD epitope to promote angiogenesis and inflammation depends in part on its flanking KGE motif. Interestingly, a subset of macrophages may represent a physiologically relevant source of this collagen epitope. Here, we define an endothelial cell mechano-signaling pathway in which a cryptic collagen epitope activates αvβ3 leading to an Src and p38 MAPK-dependent cascade that leads to nuclear accumulation of Yes-associated protein (YAP) and stimulation of endothelial cell growth. Collectively, our findings not only provide evidence for a novel mechano-signaling pathway, but also define a possible therapeutic strategy to control αvβ3 signaling by targeting a pro-angiogenic and inflammatory ligand of αvβ3 rather than the receptor itself.     

Integrin αvβ3 Requirement for Sustained Mitogen-activated Protein Kinase Activity during Angiogenesis

Angiogenesis depends on growth factors and vascular cell adhesion events. Integrins and growth factors are capable of activating the ras/MAP kinase pathway in vitro, yet how these signals influence endothelial cells during angiogenesis is unknown. Upon initiation of angiogenesis with basic fibroblast growth factor (bFGF) on the chick chorioallantoic membrane (CAM), endothelial cell mitogen-activated protein (MAP) kinase (ERK) activity was detected as early as 5 min yet was sustained for at least 20 h. The initial wave of ERK activity (5–120 min) was refractory to integrin antagonists, whereas the sustained activity (4–20 h) depended on integrin αvβ3, but not β1 integrins. Inhibition of MAP kinase kinase (MEK) during this sustained αvβ3-dependent ERK signal blocked the formation of new blood vessels while not influencing preexisting blood vessels on the CAM. Inhibition of MEK also blocked growth factor induced migration but not adhesion of endothelial cells in vitro. Therefore, angiogenesis depends on sustained ERK activity regulated by the ligation state of both a growth factor receptor and integrin αvβ3.     

A Role for the αvβ3 Integrin in the Transmigration of Monocytes

The β2 integrins and intercellular adhesion molecule-1 (ICAM-1) are important for monocyte migration through inflammatory endothelium. Here we demonstrate that the integrin αvβ3 is also a key player in this process. In an in vitro transendothelial migration assay, monocytes lacking β3 integrins revealed weak migratory ability, whereas monocytes expressing β3 integrins engaged in stronger migration. This migration could be partially blocked by antibodies against the integrin chains αL, β2, αv, or IAP, a protein functionally associated with αvβ3 integrin. Transfection of β3 integrin chain cDNA into monocytes lacking β3 integrins resulted in expression of the αvβ3 integrin and conferred on these cells an enhanced ability to transmigrate through cell monolayers expressing ICAM-1. These monocytes also engaged in αLβ2-dependent locomotion on recombinant ICAM-1 which was enhanced by αvβ3 integrin occupancy. Antibodies against IAP were able to revert this αvβ3 integrin-dependent cell locomotion to control levels. Finally, adhesion assays revealed that occupancy of αvβ3 integrin could decrease monocyte binding to ICAM-1.In conclusion, we show that αvβ3 integrin modulates αLβ2 integrin-dependent monocyte adhesion to and migration on ICAM-1. This could represent a novel mechanism to promote monocyte motility on vascular ICAM-1 and initiate subsequent transendothelial migration.     

The Thrombospondin Receptor CD47 (IAP) Modulates and Associates with α2β1 Integrin in Vascular Smooth Muscle Cells

The carboxyl-terminal domain of thrombospondin-1 enhances the migration and proliferation of smooth muscle cells. Integrin-associated protein (IAP or CD47) is a receptor for the thrombospondin-1 carboxyl-terminal cell-binding domain and binds the agonist peptide 4N1K (kRFYVVMWKk) from this domain. 4N1K peptide stimulates chemotaxis of both human and rat aortic smooth muscle cells on gelatin-coated filters. The migration on gelatin is specifically blocked by monoclonal antibodies against IAP and a β1 integrin, rather than αvβ3 as found previously for 4N1K-stimulated chemotaxis of endothelial cells on gelatin. Both human and rat smooth muscle cells displayed a weak migratory response to soluble type I collagen; however, the presence of 4N1K peptide or intact thrombospondin-1 provoked a synergistic chemotactic response that was partially blocked by antibodies to α2 and β1 integrin subunits and to IAP. A combination of antiα2 and IAP monoclonal antibodies completely blocked chemotaxis. RGD peptide and antiαvβ3 mAb were without effect. 4N1K and thrombospondin-1 did not augment the chemotactic response of smooth muscle cells to fibronectin, vitronectin, or collagenase-digested type I collagen. Complex formation between α2β1 and IAP was detected by the coimmunoprecipitation of both α2 and β1 integrin subunits with IAP. These data suggest that IAP can associate with α2β1 integrin and modulate its function.     

p21-activated Kinase 4 Phosphorylation of Integrin β5 Ser-759 and Ser-762 Regulates Cell Migration

Modulation of integrin αvβ5 regulates vascular permeability, angiogenesis, and tumor dissemination. In addition, we previously found a role for p21-activated kinase 4 (PAK4) in selective regulation of integrin αvβ5-mediated cell motility (Zhang, H., Li, Z., Viklund, E. K., and Strömblad, S. (2002) J. Cell Biol. 158, 1287–1297). This report focuses on the molecular mechanisms of this regulation. We here identified a unique PAK4-binding membrane-proximal integrin β5-SERS-motif involved in controlling cell attachment and migration. We also mapped the integrin β5-binding site within PAK4. We found that PAK4 binding to integrin β5 was not sufficient to promote cell migration, but that PAK4 kinase activity was required for PAK4 promotion of cell motility. Importantly, PAK4 specifically phosphorylated the integrin β5 subunit at Ser-759 and Ser-762 within the β5-SERS-motif. Point mutation of these two serine residues abolished the PAK4-induced cell migration, indicating a functional role for these phosphorylations in migration. Our results may give important leads to the functional regulation of integrin αvβ5, with implications for vascular permeability, angiogenesis, and cancer dissemination.     

Enhanced delivery of antisense oligonucleotides with fluorophore-conjugated PAMAM dendrimers

PAMAM dendrimers are cationic polymers that have been used for the delivery of genes and oligonucleotides to cells. However, little is known about the behavior of dendrimer–nucleic acid complexes once they reach the cell interior. To pursue this issue, we prepared dendrimers conjugated with the fluorescent dye Oregon green 488. These were used in conjunction with oligonucleotides labeled with a red (TAMRA) fluorophore in order to visualize the sub-cellular distribution of the dendrimer–oligonucleotide complex and of its components by two-color digital fluorescence microscopy. The 2′-O-methyl antisense oligonucleotide sequence used in these studies was designed to correct splicing at an aberrant intron inserted into a luciferase reporter gene; thus effective delivery of the antisense agent results in the expression of the reporter gene product. The dendrimer–oligonucleotide complex remained associated during the process of uptake into vesicular compartments and eventual entry into the nucleus. Since the pharmacological activity of the antisense compound was manifest under these conditions, it suggests that the dendrimer–oligonucleotide complex is functionally active. A surprising result of these studies was that the Oregon green 488-conjugated dendrimer was a much better delivery agent for antisense compounds than unmodified dendrimer. This suggests that coupling of relatively hydrophobic small molecules to PAMAM dendrimers may provide a useful means of enhancing their capabilities as delivery agents for nucleic acids.     

Growth Factor–dependent Activation of αvβ3 Integrin in Normal Epithelial Cells: Implications for Tumor Invasion

Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix ligands. To investigate whether cytokines produced during stromal infiltration of carcinoma cells activate nonfunctional epithelial integrins, a cellular system of human thyroid clones derived from normal glands (HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, αvβ3 integrin was diffused all over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in HTU-34 cells, αvβ3 was clustered at focal contacts (FCs) and mediated firm attachment and spreading. αvβ3 recruitment at FCs and ligand-binding activity, essentially identical to those of HTU-34, occurred in HTU-5 cells upon treatment with hepatocyte growth factor/scatter factor (HGF/SF). The HTU-34 clone secreted HGF/SF and its receptor was constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for αvβ3 activated state. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted αvβ3 enrichment at FCs and impaired adhesion. Accordingly, activation of αvβ3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF. These results provide the first example of a growth factor–driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.     

Apoptosis Signal-Regulating Kinase 1 Is Involved in WISP-1–Promoted Cell Motility in Human Oral Squamous Cell Carcinoma Cells

Oral squamous cell carcinoma (OSCC) has a tendency to migrate and metastasize. WNT1-inducible signaling pathway protein 1 (WISP-1) is a cysteine-rich protein that belongs to the Cyr61, CTGF, Nov (CCN) family of matrix cellular proteins. The effect of WISP-1 on human OSCC cells, however, is unknown. Here, we showed that WISP-1 increased cell migration and intercellular adhesion molecule-1 (ICAM-1) expression in OSCC cells. Pretreatment of cells with integrin αvβ3 monoclonal antibody (mAb) significantly abolished WISP-1–induced cell migration and ICAM-1 expression. On the other hand, WISP-1–mediated cell motility and ICAM-1 upregulation were attenuated by ASK1, JNK, and p38 inhibitor. Furthermore, WISP-1 also enhanced activator protein 1 (AP-1) activation, and the integrin αvβ3 mAb, and ASK1, JNK, and p38 inhibitors reduced WISP-1–mediated AP-1 activation. Moreover, WISP-1 and ICAM-1 expression correlated with the tumor stage of patients with OSCC. Our results indicate that WISP-1 enhances the migration of OSCC cells by increasing ICAM-1 expression through the αvβ3 integrin receptor and the ASK1, JNK/p38, and AP-1 signal transduction pathways.     

Muscle β1D Integrin Reinforces the Cytoskeleton–Matrix Link: Modulation of Integrin Adhesive Function by Alternative Splicing

Expression of muscle-specific β1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, β1 integrin-minus cells leads to their phenotypic conversion. β1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility compared with their β1A-expressing counterparts. The transfected β1D is targeted to focal adhesions and efficiently displaces the endogenous β1A and αvβ3 integrins from the sites of cell–matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in β1D transfectants. Whereas a significant part of cellular β1A integrin is extractable in digitonin, the majority of the transfected β1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton. Increased interaction of β1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin. In contrast, β1A interacts more strongly with α-actinin, than β1D. Inside-out driven activation of the β1D ectodomain increases ligand binding and fibronectin matrix assembly by β1D transfectants. Phenotypic effects of β1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of β1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton– matrix link in muscle cells. This reflects the major role for β1D integrin in muscle, where extremely stable association is required for contraction.     

Cryo-Electron Microscopy Structure of an Adenovirus-Integrin Complex Indicates Conformational Changes in both Penton Base and Integrin

A structure of adenovirus type 12 (HAdV12) complexed with a soluble form of integrin αvβ5 was determined by cryo-electron microscopy (cryoEM) image reconstruction. Subnanometer resolution (8 Å) was achieved for the icosahedral capsid with moderate resolution (27 Å) for integrin density above each penton base. Modeling with αvβ3 and α_IIbβ3 crystal structures indicates that a maximum of four integrins fit over the pentameric penton base. The close spacing (∼60 Å) of the RGD protrusions on penton base precludes integrin binding in the same orientation to neighboring RGD sites. Flexible penton-base RGD loops and incoherent averaging of bound integrin molecules explain the moderate resolution observed for the integrin density. A model with four integrins bound to a penton base suggests that integrin might extend one RGD-loop in the direction that could induce a conformational change in the penton base involving clockwise untwisting of the pentamer. A global conformational change in penton base could be one step on the way to the release of Ad vertex proteins during cell entry. Comparison of the cryoEM structure with bent and extended models for the integrin ectodomain reveals that integrin adopts an extended conformation when bound to the Ad penton base, a multivalent viral ligand. These findings shed further light on the structural basis of integrin binding to biologically relevant ligands, as well as on the molecular events leading to HAdV cell entry.A growing number of viruses have been identified as using one of the 24 types of integrin heterodimers as a receptor for cell entry (32). Integrins are cell surface molecules involved in the regulation of adhesion, migration, growth, and differentiation (11). The large multidomained extracellular segments of α and β integrin subunits bind a variety of ligands, including viral ligands, while the smaller intracellular domains interact with cytoskeletal proteins (Fig. ​(Fig.1A).1A). These extracellular and intracellular interactions facilitate bidirectional signaling, with the initiating events occurring either outside of the cell (outside-in signaling) or within the cell (inside-out signaling) (24). Integrin clustering has been established as having an important role in outside-in signaling (9, 19, 20, 44). Clustering results in the formation of focal adhesions, which are organized intracellular complexes, that facilitate downstream signaling cascades within the cell (24).Open in a separate windowFIG. 1.Integrin domains and conformations. (A) Structural domains of integrin αv and β chains, including the extracellular domains, transmembrane-spanning regions, and small cytoplasmic domains, shown in extended schematic forms. The domains are represented as 10Å-resolution density maps based on crystallographic coordinates. The membrane is represented by a gray bar. (Modified from Stewart and Nemerow (32) and reprinted with permission from Elsevier.) (B) Models for soluble αvβ5 integrin with Fos/Jun dimerization domains. Each chain has a six residue glycine-rich linker between the ectodomain and the Fos or Jun dimerization domain. The model of a bent integrin conformation (left) was built as a composite of αvβ3 integrin crystal structures, PDB-IDs 1L5G and 1U8C (42, 43), and the crystal structure of c-Fos/c-Jun bound to DNA, PDB-ID 1FOS (6). The model of an extended integrin conformation (right) is similar to the extended model docked into the HAdV12/αvβ5 cryo structure (Fig. ​(Fig.8B8B).Studies of adenovirus (Ad) interactions with αv integrins provided some of the first evidence of the virus-induced signaling events (13, 14). The Ad penton base capsid protein, which sits at the 12 vertices of the icosahedral capsid, has five prominent Arg-Gly-Asp (RGD) containing loops that are flexible and protrude from the viral surface (31, 48). Receptor-mediated endocytosis of Ad is stimulated by interaction of the RGD-containing penton base with αvβ3 and αvβ5 integrins (34). This interaction leads to receptor clustering, followed by tyrosine phosphorylation/activation of focal adhesion kinase, as well as activation of p130CAS, phosphatidylinositol 3-OH-kinase, and the Rho family of small GTPases, and subsequent actin polymerization and Ad internalization (32). Integrin signaling events also lead to production of proinflammatory cytokines (23) and may result in increased survival of certain host cells through subsequent signaling to protein kinase B (AKT) (25).Multiple studies indicate that after interaction with an RGD-containing ligand a straightening of the integrin extracellular domains occurs, leading to the “extension” or “switchblade” model for integrin activation (16, 45). In the extension model the headpiece domains, which are closest to the RGD interaction site, have a “closed” conformation in the low-affinity, unliganded state. This state is characterized by the close proximity of the α and β subunits at the “knees” or midpoints of the extracellular segments. In contrast, the high-affinity, ligand-bound state in the extension model is distinguished by an “open” headpiece conformation with separation at the knees of the extracellular segments. The location of the RGD binding site between the α-subunit β-propellor and the β-subunit I domain was first visualized in the crystal structure of the αvβ3 extracellular segment with a bound RGD peptide (43). In this structure the RGD site is folded back toward the membrane, and the integrin is in a closed conformation. The closed conformation has also been observed in crystal structures of the αvβ3 ectodomain without an RGD peptide (41) and the α_IIbβ3 ectodomain (47).The open integrin conformation has been characterized as having a large separation of up to ∼70 Å between the knees of α and β subunits (16). Four slightly different open headpiece conformations were observed in crystal structures of the α_IIbβ3 headpiece with bound fibrinogen-mimetic therapeutics (38). These structures show that the change from a closed to an open headpiece conformation is accompanied by a piston-like motion of helix α7 in the β-chain I domain and a large swing of the β-chain hybrid domain of up to 69°, as well as extension and separation of the two integrin chains. Comparison of the available αvβ3 and α_IIbβ3 crystal structures is providing information on the interdomain angle variation and flexibility between domains (47).One aspect of the extension model is that separation of the C-terminal, intracellular portions of the α and β subunits leads to inside-out activation. This concept is supported by nuclear magnetic resonance structures of the cytoplasmic tails of α_IIbβ3 showing that the membrane-proximal helices engage in a weak interaction that can be disrupted by constitutively activating mutations or by talin, a protein found in high concentrations in focal adhesions (33). The concept that the integrin α and β subunits must also separate during outside-in signaling is supported by a study involving a disulfide-bonded mutant of α_IIbβ3 integrin (46). When the α and β subunits are linked in the vicinity of the transmembrane helices the mutant α_IIbβ3 is still able to bind ligand, mediate adhesion, and undergo antibody-induced clustering. However, the disulfide-bonded mutant exhibits defects in focal adhesion formation and focal adhesion kinase activation. Reduction of the disulfide bond or single cysteine mutants rescues signaling.A competing model for integrin activation, called the “deadbolt” model, proposes only small conformational changes in the integrin β-chain I domain upon RGD binding (2). This model is based on crystal structures of the αvβ3 ectodomain with or without an RGD peptide (41, 43). Both of these αvβ3 structures reveal a bent integrin conformation with a closed headpiece conformation. However, the RGD peptide was soaked into a preformed crystal of αvβ3 and crystal contacts may have prevented conformational changes.There are relatively few and only moderate resolution structures of virus-integrin complexes. A moderate resolution cryoEM structure has been determined for the Picornavirus echovirus 1 (EV1) in complex with the I domain of the α2 integrin subunit (39). Docking of crystal structures of EV1 and the α2 I domain into the cryoEM density indicates that the I domain binds within a canyon on the surface of EV1 and that five integrins could potentially bind at one vertex of the icosahedral capsid. Confocal fluorescence microscopy experiments indicated that EV1 causes integrin clustering on human osteosarcoma cells stably transfected with α2 integrin. However, it could not be determined whether the bound integrins were in the inactive (bent) or active (extended) conformation.Moderate resolution (∼21 Å) cryoEM structures of Ad type 2 (HAdV2) and HAdV12 in complex with a soluble form of αvβ5 integrin revealed a ring of integrin density over each penton base capsid protein (5). Better-defined integrin density was observed in the HAdV12/integrin complex, supporting the idea suggested from sequence alignments that the RGD loop of the HAdV12 penton base is shorter and less flexible than that of HAdV2. This study also suggested that the precise spatial arrangement of the five RGD protrusions on the penton base might promote integrin clustering, which may lead to the intracellular signaling events required for virus internalization into a host cell. A similar spacing of RGD-containing integrin-binding sites around the fivefold axis of icosahedral virions has been noted for Ad, foot-and-mouth disease virus, and coxsackievirus A9 (32).We present here a significantly higher-resolution cryoEM structure of HAdV12 complexed with soluble αvβ5 that provides insight into the Ad-integrin interaction. The resolution of the icosahedral capsid portion of the Ad-integrin complex was improved to 8 Å, and the capsid shows clearly resolved α-helices, which allows accurate docking of the penton base crystal structure within the cryoEM density. The resolution of the integrin density is more moderate due to flexibility of the RGD-containing surface loop of penton base and incoherent averaging of integrin heterodimers. Nevertheless, modeling studies with available integrin crystal structures have enabled us to distinguish between a bent or extended conformation (Fig. ​(Fig.1B)1B) when αvβ5 binds to the multivalent ligand presented by the Ad penton base. The cryoEM structural analysis also indicates that integrin induces a conformational change in penton base.     

The 5' stem-loop regulates expression of collagen alpha1(I) mRNA in mouse fibroblasts cultured in a three-dimensional matrix

The stability of collagen α1(I) mRNA is regulated by its 5′ stem–loop, which binds a cytoplasmic protein in a cap-dependent manner, and its 3′-untranslated region (UTR), which binds αCP. When cultured in a three-dimensional gel composed of type I collagen, mouse fibroblasts had decreased collagen α1(I) mRNA steady-state levels, which resulted from a decreased mRNA half-life. In cells cultured in gel, hybrid mouse–human collagen α1(I) mRNA with a wild-type 5′ stem–loop decayed faster than the same mRNA with a mutated stem–loop. When the 5′ stem–loop was placed in a heterologous mRNA, the mRNA accumulated to a lower level in cells grown in gel than in cells grown on plastic. This suggests that the 5′ stem–loop down-regulates collagen α1(I) mRNA. Protein binding to the 5′ stem–loop was reduced in cells grown in gel, which was associated with destabilization of the collagen α1(I) mRNA. In addition to the binding of a cytoplasmic protein, there was also a nuclear binding activity directed to the collagen α1(I) 5′ stem–loop. The nuclear binding was increased in cells grown in gel, suggesting that it may negatively regulate expression of collagen α1(I) mRNA. Binding of αCP, a protein involved in stabilization of collagen α1(I) mRNA, was unchanged by the culture conditions.     

Syndecan-1 and Syndecan-4 Capture Epidermal Growth Factor Receptor Family Members and the α3β1 Integrin Via Binding Sites in Their Ectodomains: NOVEL SYNSTATINS PREVENT KINASE CAPTURE AND INHIBIT α6β4-INTEGRIN-DEPENDENT EPITHELIAL CELL MOTILITY*

The α6β4 integrin is known to associate with receptor tyrosine kinases when engaged in epithelial wound healing and in carcinoma invasion and survival. Prior work has shown that HER2 associates with α6β4 integrin and syndecan-1 (Sdc1), in which Sdc1 engages the cytoplasmic domain of the β4 integrin subunit allowing HER2-dependent motility and carcinoma cell survival. In contrast, EGFR associates with Sdc4 and the α6β4 integrin, and EGFR-dependent motility depends on cytoplasmic engagement of β4 integrin with Sdc4. However, how HER2 and EGFR assimilate into a complex with the syndecans and integrin, and why kinase capture is syndecan-specific has remained unknown. In the present study, we demonstrate that HER2 is captured via a site, comprised of amino acids 210–240, in the extracellular domain of human Sdc1, and EGFR is captured via an extracellular site comprised of amino acids 87–131 in human Sdc4. Binding assays using purified recombinant proteins demonstrate that the interaction between the EGFR family members and the syndecans is direct. The α3β1 integrin, which is responsible for the motility of the cells, is captured at these sites as well. Peptides based on the interaction motifs in Sdc1 and Sdc4, called synstatins (SSTN_210–240 and SSTN_87–131) competitively displace the receptor tyrosine kinase and α3β1 integrin from the syndecan with an IC₅₀ of 100–300 nm. The syndecans remain anchored to the α6β4 integrin via its cytoplasmic domain, but the activation of cell motility is disrupted. These novel SSTN peptides are potential therapeutics for carcinomas that depend on these HER2- and EGFR-coupled mechanisms for their invasion and survival.