The α5β1 Integrin Mediates Elimination of Amyloid-β Peptide and Protects Against Apoptosis

The amyloid-β peptide (Aβ) can mediate cell attachment by binding to β1 integrins through an arg-his-asp sequence. We show here that the α5β1 integrin, a fibronectin receptor, is an efficient binder of Aβ, and mediates cell attachment to nonfibrillar Aβ. Cells engineered to express α5β1 internalized and degraded more added Aβ1-40 than did α5β1-negative control cells. Deposition of an insoluble Aβ1-40 matrix around the α5β1-expressing cells was reduced, and the cells showed less apoptosis than the control cells. Thus, the α5β1 integrin may protect against Aβ deposition and toxicity, which is a course of Alzheimer's disease lesions.     

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.     

Linking Integrin α6β4-based Cell Adhesion to the Intermediate Filament Cytoskeleton: Direct Interaction between the β4 Subunit and Plectin at Multiple Molecular Sites

Recent studies with patients suffering from epidermolysis bullosa simplex associated with muscular dystrophy and the targeted gene disruption in mice suggested that plectin, a versatile cytoskeletal linker and intermediate filament-binding protein, may play an essential role in hemidesmosome integrity and stabilization. To define plectin's interactions with hemidesmosomal proteins on the molecular level, we studied its interaction with the uniquely long cytoplasmic tail domain of the β₄ subunit of the basement membrane laminin receptor integrin α₆β₄ that has been implicated in connecting the transmembrane integrin complex with hemidesmosome-anchored cytokeratin filaments. In vitro binding and in vivo cotransfection assays, using recombinant mutant forms of both proteins, revealed their direct interaction via multiple molecular domains. Furthermore, we show in vitro self-interaction of integrin β₄ cytoplasmic domains, as well as disruption of intermediate filament network arrays and dislocation of hemidesmosome-associated endogenous plectin upon ectopic overexpression of this domain in PtK2 and/or 804G cells. The close association of plectin molecules with hemidesmosomal structures and their apparent random orientation was indicated by gold immunoelectron microscopy using domain-specific antibodies. Our data support a model in which plectin stabilizes hemidesmosomes, via directly interlinking integrin β₄ subunits and cytokeratin filaments.     

Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail

Platelet agonists increase the affinity state of integrin α_IIbβ₃, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. β₃-Endonexin is a novel 111–amino acid protein that binds selectively to the β₃ tail. Since β₃-endonexin is present in platelets, we asked whether it can affect α_IIbβ₃ function. When β₃-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor α_IIbβ₃ affinity state in transfected cells by flow cytometry. Cells transfected with GFP and α_IIbβ₃ bound little or no PAC1. However, those transfected with GFP/β₃-endonexin and α_IIbβ₃ bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β₃-endonexin did not affect levels of surface expression of α_IIbβ₃ nor did it modulate the affinity of an α_IIbβ₃ mutant that is defective in binding to β₃-endonexin. Affinity modulation of α_IIbβ₃ by GFP/β₃-endonexin was inhibited by coexpression of either a monomeric β₃ cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β₃-endonexin can modulate the affinity state of α_IIbβ₃ in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein–protein interactions at the level of the cytoplasmic tails of α_IIbβ₃.     

Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

Chondroadherin (the 36-kD protein) is a leucine-rich, cartilage matrix protein known to mediate adhesion of isolated chondrocytes. In the present study we investigated cell surface proteins involved in the interaction of cells with chondroadherin in cell adhesion and by affinity purification. Adhesion of bovine articular chondrocytes to chondroadherin-coated dishes was dependent on Mg²⁺ or Mn²⁺ but not Ca²⁺. Adhesion was partially inhibited by an antibody recognizing β1 integrin subunit. Chondroadherin-binding proteins from chondrocyte lysates were affinity purified on chondroadherin-Sepharose. The β1 integrin antibody immunoprecipitated two proteins with molecular mass ~110 and 140 kD (nonreduced) from the EDTA-eluted material. These results indicate that a β1 integrin on chondrocytes interacts with chondroadherin. To identify the α integrin subunit(s) involved in interaction of cells with the protein, we affinity purified chondroadherin-binding membrane proteins from human fibroblasts. Immunoprecipitation of the EDTA-eluted material from the affinity column identified α2β1 as a chondroadherin-binding integrin. These results are in agreement with cell adhesion experiments where antibodies against the integrin subunit α2 partially inhibited adhesion of human fibroblast and human chondrocytes to chondroadherin. Since α2β1 also is a receptor for collagen type II, we tested the ability of different antibodies against the α2 subunit to inhibit adhesion of T47D cells to collagen type II and chondroadherin. The results suggested that adhesion to collagen type II and chondroadherin involves similar or nearby sites on the α2β1 integrin. Although α2β1 is a receptor for both collagen type II and chondroadherin, only adhesion of cells to collagen type II was found to mediate spreading.     

Modulation of In Vivo Migratory Function of α2β1 Integrin in Mouse Liver

We report herein that expression of α2β1 integrin increased human erythroleukemia K562 transfectant (KX2C2) cell movement after extravasation into liver parenchyma. In contrast, a previous study demonstrated that α2β1 expression conferred a stationary phenotype to human rhabdomyosarcoma RD transfectant (RDX2C2) cells after extravasation into the liver. We therefore assessed the adhesive and migratory function of α2β1 on KX2C2 and RDX2C2 cells using a α2β1-specific stimulatory monoclonal antibody (mAb), JBS2, and a blocking mAb, BHA2.1. In comparison with RDX2C2 cells, KX2C2 were only weakly adherent to collagen and laminin. JBS2 stimulated α2β1-mediated interaction of KX2C2 cells with both collagen and laminin resulting in increases in cell movement on both matrix proteins. In the presence of Mn²⁺, JBS2-stimulated adhesion on collagen beyond an optimal level for cell movement. In comparison, an increase in RDX2C2 cell movement on collagen required a reduction in its adhesive strength provided by the blocking mAb BHA2.1. Consistent with these in vitro findings, in vivo videomicroscopy revealed that α2β1-mediated postextravasation cell movement of KX2C2 cells in the liver tissue could also be stimulated by JBS2. Thus, results demonstrate that α2β1 expression can modulate postextravasation cell movement by conferring either a stationary or motile phenotype to different cell types. These findings may be related to the differing metastatic activities of different tumor cell types.     

Integrin αvβ1 Is a Receptor for Foot-and-Mouth Disease Virus

Infection by field strains of Foot-and-mouth disease virus (FMDV) is initiated by binding to certain species of arginine-glycine-aspartic acid (RGD)-dependent integrin including alphavbeta3 and the epithelial integrin alphavbeta6. In this report we show that the integrin alphavbeta1, when expressed as a human/hamster heterodimer on transfected CHOB2 cells, is a receptor for FMDV. Virus binding and infection mediated by alphavbeta1 was inefficient in the presence of physiological concentrations of calcium and magnesium but were significantly enhanced by reagents that activate the integrin and promote ligand binding. The ability of chimeric alpha5/alphav integrin subunits, in association with the beta1 chain, to bind FMDV and mediate infection matched the ligand binding specificity of alphavbeta1, not alpha5beta1, thus providing further evidence for the receptor role of alphavbeta1. In addition, data are presented suggesting that amino acid residues near the RGD motif may be important for differentiating between the binding specificities of alphavbeta1 and alphavbeta6.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

Evidence that Distinct States of the Integrin α6β1 Interact with Laminin and an ADAM

Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin α6β1, on mouse eggs and on α6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin β). In the present study we tested the hypothesis that different states of α6β1 interact with fertilin and laminin, an extracellular matrix ligand for α6β1. Using α6-transfected cells we found that treatments (e.g., with phorbol myristate acetate or MnCl₂) that increased adhesion to laminin inhibited sperm binding. Conversely, treatments that inhibited laminin adhesion increased sperm binding. Next, we compared the ability of fluorescent beads coated with either fertilin β or with the laminin E8 fragment to bind to eggs. In Ca²⁺-containing media, fertilin β beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin β and by the α6 integrin subunit. In Ca²⁺-containing media, laminin E8 beads did not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding. Treatment of eggs with Mn²⁺ dramatically increased laminin E8 bead binding, and inhibited fertilin bead binding. Our results provide the first evidence that different states of an integrin (α6β1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).     

Use of a β1 Integrin-deficient Human T Cell to Identify β1 Integrin Cytoplasmic Domain Sequences Critical for Integrin Function

T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in β1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the β1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human β1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the β1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the β1 integrin with the activating β1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the β1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of β1 integrin structure and function in human T cells.     

Modulation of β1A Integrin Functions by Tyrosine Residues in the β1 Cytoplasmic Domain

β1A integrin subunits with point mutations of the cytoplasmic domain were expressed in fibroblasts derived from β1-null stem cells. β1A in which one or both of the tyrosines of the two NPXY motifs (Y783, Y795) were changed to phenylalanines formed active α5β1 and α6β1 integrins that mediated cell adhesion and supported assembly of fibronectin. Mutation of the proline in either motif (P781, P793) to an alanine or of a threonine in the inter-motif sequence (T788) to a proline resulted in poorly expressed, inactive β1A. Y783,795F cells developed numerous fine focal contacts and exhibited motility on a surface. When compared with cells expressing wild-type β1A or β1A with the D759A activating mutation of a conserved membrane–proximal aspartate, Y783,795F cells had impaired ability to transverse filters in chemotaxis assays. Analysis of cells expressing β1A with single Tyr to Phe substitutions indicated that both Y783 and Y795 are important for directed migration. Actin-containing microfilaments of Y783,795F cells were shorter and more peripheral than microfilaments of cells expressing wild-type β1A. These results indicate that change of the phenol side chains in the NPXY motifs to phenyl groups (which cannot be phosphorylated) has major effects on the organization of focal contacts and cytoskeleton and on directed cell motility.     

Structure of Adenovirus Complexed with Its Internalization Receptor, αvβ5 Integrin

The three-dimensional structure of soluble recombinant integrin alphavbeta5 bound to human adenovirus types 2 and 12 (Ad2 and -12) has been determined at approximately 21-A resolution by cryoelectron microscopy (cryo-EM). The alphavbeta5 integrin is known to promote Ad cell entry. Cryo-EM has shown that the integrin-binding RGD (Arg-Gly-Asp) protrusion of the Ad2 penton base protein is highly mobile (P. L. Stewart, C. Y. Chiu, S. Huang, T. Muir, Y. Zhao, B. Chait, P. Mathias, and G. R. Nemerow, EMBO J. 16:1189-1198, 1997). Sequence analysis indicated that the Ad12 RGD surface loop is shorter than that of Ad2 and probably less flexible, hence more suitable for structural characterization of the Ad-integrin complex. The cryo-EM structures of the two virus-receptor complexes revealed a ring of integrin density above the penton base of each virus serotype. As expected, the integrin density in the Ad2 complex was diffuse while that in the Ad12 complex was better defined. The integrin consists of two discrete subdomains, a globular domain with an RGD-binding cleft approximately 20 A in diameter and a distal domain with extended, flexible tails. Kinetic analysis of Ad2 interactions with alphavbeta5 indicated approximately 4.2 integrin molecules bound per penton base at close to saturation. These results suggest that the precise spatial arrangement of five RGD protrusions on the penton base promotes integrin clustering and the signaling events required for virus internalization.     

α3β1 Integrin Is Required for Normal Development of the Epidermal Basement Membrane

Integrins α3β1 and α6β4 are abundant receptors on keratinocytes for laminin-5, a major component of the basement membrane between the epidermis and the dermis in skin. These integrins are recruited to distinct adhesion structures within keratinocytes; α6β4 is present in hemidesmosomes, while α3β1 is recruited into focal contacts in cultured cells. To determine whether differences in localization reflect distinct functions of these integrins in the epidermis, we studied skin development in α3β1-deficient mice. Examination of extracellular matrix by immunofluorescence microscopy and electron microscopy revealed regions of disorganized basement membrane in α3β1-deficient skin. Disorganized matrix was first detected by day 15.5 of embryonic development and became progressively more extensive as development proceeded. In neonatal skin, matrix disorganization was frequently accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa. Consistent with this notion, primary keratinocytes from α3β1-deficient skin adhered to laminin-5 through α6 integrins. However, α3β1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for α3β1 and indicating distinct roles for α3β1 and α6β4. Our findings support a novel role for α3β1 in establishment and/or maintenance of basement membrane integrity, while α6β4 is required for stable adhesion of the epidermis to the basement membrane through hemidesmosomes.     

Function of the Tetraspanin CD151–α6β1 Integrin Complex during Cellular Morphogenesis

Upon plating on basement membrane Matrigel, NIH3T3 cells formed an anastomosing network of cord-like structures, inhibitable by anti-alpha6beta1 integrin antibodies. For NIH3T3 cells transfected with human CD151 protein, the formation of a cord-like network was also inhibitable by anti-CD151 antibodies. Furthermore, CD151 and alpha6beta1 were physically associated within NIH3T3 cells. On removal of the short 8-amino acid C-terminal CD151 tail (by deletion or exchange), exogenous CD151 exerted a dominant negative effect, as it almost completely suppressed alpha6beta1-dependent cell network formation and NIH3T3 cell spreading on laminin-1 (an alpha6beta1 ligand). Importantly, mutant CD151 retained alpha6beta1 association and did not alter alpha6beta1-mediated cell adhesion to Matrigel. In conclusion, the CD151-alpha6beta1 integrin complex acts as a functional unit that markedly influences cellular morphogenesis, with the CD151 tail being of particular importance in determining the "outside-in" functions of alpha6beta1-integrin that follow ligand engagement. Also, antibodies to alpha6beta1 and CD151 inhibited formation of endothelial cell cord-like networks, thus pointing to possible relevance of CD151-alpha6beta1 complexes during angiogenesis.     

Neuropilin-1/GIPC1 Signaling Regulates α5β1 Integrin Traffic and Function in Endothelial Cells

Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A). Nevertheless, Nrp1 null embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes α5β1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with α5β1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active α5β1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with α5β1, and the associated motor myosin VI (Myo6) support active α5β1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active α5β1. Nrp1 modulation of α5β1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice.     

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α’-chain (α’NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α’NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.     

The Arg-Gly-Asp Motif in the Cell Adhesion Molecule L1 Promotes Neurite Outgrowth via Interaction with the αvβ3 Integrin

The cell adhesion molecule L1 is a potent inducer of neurite outgrowth and it has been implicated in X-linked hydrocephalus and related neurological disorders. To investigate the mechanisms of neurite outgrowth stimulated by L1, attempts were made to identify the neuritogenic sites in L1. Fusion proteins containing different segments of the extracellular region of L1 were prepared and different neuronal cells were assayed on substrate-coated fusion proteins. Interestingly, both immunoglobulin (Ig)-like domains 2 and 6 (Ig2, Ig6) promoted neurite outgrowth from dorsal root ganglion cells, whereas neural retinal cells responded only to Ig2. L1 Ig2 contains a previously identified homophilic binding site, whereas L1 Ig6 contains an Arg-Gly-Asp (RGD) sequence. The neuritogenic activity of Ig6 was abrogated by mutations in the RGD site. The addition of RGD-containing peptides also inhibited the promotion of neurite outgrowth from dorsal root ganglion cells by glutathione S-transferase-Ig6, implicating the involvement of an integrin. The monoclonal antibody LM609 against α_vβ₃ integrin, but not an anti-β₁ antibody, inhibited the neuritogenic effects of Ig6. These data thus provide the first evidence that the RGD motif in L1 Ig6 is capable of promoting neurite outgrowth via interaction with the α_vβ₃ integrin on neuronal cells.     

Force Measurements of the α5β1 Integrin–Fibronectin Interaction

The interaction of the α₅β₁ integrin and its ligand, fibronectin (FN), plays a crucial role in the adhesion of cells to the extracellular matrix. An important intrinsic property of the α₅β₁/FN interaction is the dynamic response of the complex to a pulling force. We have carried out atomic force microscopy measurements of the interaction between α₅β₁ and a fibronectin fragment derived from the seventh through tenth type III repeats of FN (i.e., FN7-10) containing both the arg-gly-asp (RGD) sequence and the synergy site. Direct force measurements obtained from an experimental system consisting of an α₅β₁ expressing K562 cell attached to the atomic force microscopy cantilever and FN7-10 adsorbed on a substrate were used to determine the dynamic response of the α₅β₁/FN7-10 complex to a pulling force. The experiments were carried out over a three-orders-of-magnitude change in loading rate and under conditions that allowed for detection of individual α₅β₁/FN7-10 interactions. The dynamic rupture force of the α₅β₁/FN7-10 complex revealed two regimes of loading: a fast loading regime (>10,000 pN/s) and a slow loading regime (<10,000 pN/s) that characterize the inner and outer activation barriers of the complex, respectively. Activation by TS2/16 antibody increased both the frequency of adhesion and elevated the rupture force of the α₅β₁/wild type FN7-10 complex to higher values in the slow loading regime. In experiments carried out with a FN7-10 RGD deleted mutant, the force measurements revealed that both inner and outer activation barriers were suppressed by the mutation. Mutations to the synergy site of FN, however, suppressed only the outer barrier activation of the complex. For both the RGD and synergy deletions, the frequency of adhesion was less than that of the wild type FN7-10, but was increased by integrin activation. The rupture force of these mutants was only slightly less than that of the wild type, and was not increased by activation. These results suggest that integrin activation involved a cooperative interaction with both the RGD and synergy sites.     

β1 Integrin Is Essential for Teratoma Growth and Angiogenesis

Teratomas are benign tumors that form after ectopic injection of embryonic stem (ES) cells into mice and contain derivatives of all primitive germ layers. To study the role of β1 integrin during teratoma formation, we compared teratomas induced by normal and β1-null ES cells. Injection of normal ES cells gave rise to large teratomas. In contrast, β1-null ES cells either did not grow or formed small teratomas with an average weight of <5% of that of normal teratomas. Histological analysis of β1-null teratomas revealed the presence of various differentiated cells, however, a much lower number of host-derived stromal cells than in normal teratomas. Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas, diffusely deposited in β1-null teratomas. Basement membranes were present but with irregular shape and detached from the cell surface.

Normal teratomas had large blood vessels with a smooth inner surface, containing both host- and ES cell–derived endothelial cells. In contrast, β1-null teratomas had small vessels that were loosely embedded into the connective tissue. Furthermore, endothelial cells were always of host-derived origin and formed blood vessels with an irregular inner surface. Although β1- deficient endothelial cells were absent in teratomas, β1-null ES cells could differentiate in vitro into endothelial cells. The formation of a complex vasculature, however, was significantly delayed and of poor quality in β1-null embryoid bodies. Moreover, while vascular endothelial growth factor induced proliferation of endothelial cells as well as an extensive branching of blood vessels in normal embryoid bodies, it had no effect in β1-null embryoid bodies.

     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.