Fibronectin on target cells attenuates natural cytotoxicity of NK cells via myeloid immune checkpoint ILT3/LILRB4/gp49B.

Natural killer (NK) cells play pivotal roles in innate immunity as well as in anti-tumor responses via natural killing, while their activity is tightly regulated by cell-surface inhibitory receptors. Immunoglobulin-like transcript 3/leukocyte immunoglobulin-like receptor B4 (ILT3/LILRB4, also known as gp49B in mice) is an inhibitory receptor expressed on activated NK cells as well as myeloid-lineage cells. The common physiologic ligand of human LILRB4 and gp49B was identified very recently as fibronectin, particularly the N-terminal 30 kDa domain (FN30). We hypothesized that LILRB4 could bind fibronectin on target cells in trans together with integrins, classical fibronectin receptors, in cis and deliver an inhibitory signal in NK cells, leading to attenuated natural killing. Flow cytometric and confocal microscopic analyses of NK cell-surface gp49B and integrins suggested that these novel and classical fibronectin receptors, respectively, co-engage fibronectin immobilized on a culture plate. Biochemical analyses indicated that tyrosine phosphorylation of spleen tyrosine kinase was augmented in gp49B-deficient NK cells upon binding to the immobilized fibronectin. While surface fibronectin-poor YAC-1 cells were evenly sensitive as to natural killing of both gp49B-positive and -negative NK cells, the killing of fibronectin-rich Lewis lung carcinoma cells, but not the FN30-knockout cells, was augmented among gp49B-deficient NK cells. These results suggest that the natural cytotoxicity of NK cells is negatively regulated through LILRB4/gp49B sensing fibronectin on target cells, which sheds light on the unexpected role of LILRB4 and fibronectin as a potential attenuator of NK cell cytotoxicity in the tumor microenvironment.

Co-localization of Fibronectin Receptors LILRB4/gp49B and Integrin on Dendritic Cell Surface.

A myeloid immune checkpoint, leukocyte immunoglobulin-like receptor (LILR) B4 (B4, also known as ILT3/CD85k in humans and gp49B in mice) is expressed on dendritic cells (DCs). However, a mode of regulation of DCs by B4/gp49B is not identified yet in relation to the ligand(s) as well as to the counteracting, activation-type receptor. Our recent identification of the physiological/pathological ligand for B4/gp49B as the fibronectin (FN) N-terminal 30-kDa domain poses the question of the relationship between B4/gp49B and a classical FN receptor/cellular activator, integrin, on DCs. Here we showed that FN is not constitutively tethered on the surface of bone marrow-derived cultured DCs (BMDCs) or splenic DCs, even though the FN receptor integrin and gp49B are co-expressed on these cells. Confocal laser scanning microscopic analysis, however, revealed weak correlation of fluorescent signals between gp49B and integrin ß1, suggesting their partial co-localization on the BMDC surface even in the absence of FN. We found that the plating of BMDCs onto immobilized FN induced tyrosine phosphorylation of focal adhesion kinase (FAK) and spleen tyrosine kinase (Syk). In the absence of gp49B, while the FAK phosphorylation level was virtually unchanged, that of phosphorylation of Syk was markedly augmented. These results suggested that the immobilized FN induced a crosstalk between gp49B and integrin in terms of the intracellular signaling of BMDCs, in which gp49B suppressed the integrin-mediated pro-inflammatory cascade. Our observations may provide a clue for elucidating the mechanism of the therapeutic efficacy of B4/gp49B blocking in autoimmune disease and cancer.

[Expression and significance of integrin α5ß1 and fibronectin in atherosclerotic plaques from autopsy specimens].

Objective: To investigate the expression of integrin α5ß1 and fibronectin in the human aorta and coronary artery, and their effects in the development of atherosclerosis. Methods: One hundred and twenty autopsy aorta and coronary artery specimens were collected, and the expression of CD68, actin, integrin α5ß1 and fibronectin was detected by immunohistochemical staining. Atherosclerotic plaques were located by CD68 and actin staining, and the degree of coronary artery stenosis was determined by elastic fiber staining and NIH Scion Image(60.1) software. The coronary artery tissues were divided into groups A (0-25%); B (26%-50%); C (51%-75%) and D (76%-100%) according to the degree of stenosis. Results: Integrin α5ß1 showed cytoplasmic expression in endothelium, foam cells, monocytes, smooth muscle cells and adjacent tissue around calcification. In both the aorta and coronary artery, integrin α5ß1 expression was stronger in the smooth muscle cells in the internal elastic lamina than in the tunica. The expression intensity in coronary artery smooth muscle decreased with increasing degree of coronary artery stenosis. Fibronectin showed cytoplasmic expression in foam cells, monocytes, smooth muscle cells of the internal elastic lamina and adjacent tissue around calcification. There was positive correlation of fibronectin and integrin α5ß1 expression in smooth muscle cells and adjacent tissue around calcification. Conclusions: In the development of atherosclerosis, integrin α5ß1 and fibronectin may participate in the regulating the migration of smooth muscle cells to the intima, and promote the formation of local calcification of atherosclerotic plaques. But integrin α5ß1 is not involved in the late stage of atherosclerosis with increasing coronary artery stenosis.

Endothelial alpha5 and alphav integrins cooperate in remodeling of the vasculature during development.

Integrin cell adhesion receptors and fibronectin, one of their extracellular matrix ligands, have been demonstrated to be important for angiogenesis using functional perturbation studies and complete knockout mouse models. Here, we report on the roles of the alpha5 and alphav integrins, which are the major endothelial fibronectin receptors, in developmental angiogenesis. We generated an integrin alpha5-floxed mouse line and ablated alpha5 integrin in endothelial cells. Unexpectedly, endothelial-specific knockout of integrin alpha5 has no obvious effect on developmental angiogenesis. We provide evidence for genetic interaction between mutations in integrin alpha5 and alphav and for overlapping functions and compensation between these integrins and perhaps others. Nonetheless, in embryos lacking both alpha5 and alphav integrins in their endothelial cells, initial vasculogenesis and angiogenesis proceed normally, at least up to E11.5, including the formation of apparently normal embryonic vasculature and development of the branchial arches. However, in the absence of endothelial alpha5 and alphav integrins, but not of either alone, there are extensive defects in remodeling of the great vessels and heart resulting in death at ~E14.5. We also found that fibronectin assembly is somewhat affected in integrin alpha5 knockout endothelial cells and markedly reduced in integrin alpha5/alphav double-knockout endothelial cell lines. Therefore, neither alpha5 nor alphav integrins are required in endothelial cells for initial vasculogenesis and angiogenesis, although they are required for remodeling of the heart and great vessels. These integrins on other cells, and/or other integrins on endothelial cells, might contribute to fibronectin assembly and vascular development.

Interferon-γ inhibits integrin-mediated extracellular signal-regulated kinase activation stimulated by fibronectin binding in thyroid cells.

Hashimoto's thyroiditis (HT) is an autoimmune disorder characterized by the presence of specific antibodies and by a lymphocytic infiltration of the thyroid secreting inflammatory cytokines. Macrophages, lymphocytes, and cytokines play a pivotal role in both development and progression of Th1-mediated autoimmune diseases, and a direct role in the destruction of thyroid follicles and follicular cell function in autoimmune thyroiditis. Integrins are integral membrane receptors involved in cell-extra-cellular matrix (ECM) interaction with both structural and signaling functions. The integrin- ECM interaction is necessary for the correct function and survival of thyroid follicular cells. The purpose of this study was to determine the effect of cytokine stimulation on integrin expression and signaling in the thyroid cell. Primary cultures from normal thyroids were treated with interferon-γ (IFN-γ), INF-α, tumor necrosis factor-α, interleukin 1a or these cytokines all together. Integrin expression, cell adhesion to fibronectin (FN) and FN-stimulated extracellular signal-regulated kinase (ERK) phosphorylation were determined after cytokine treatment. IFN-γ and IFN-α were the most effective, reducing the expression of the integrin αvß3 and slightly increasing the α3ß1. Cell treatment with IFN-γ strongly impaired cell adhesion to FN. At the same time, the treatment with IFN-γ dramatically inhibited the stimulation of ERK phosphorylation induced by cell adhesion to FN. In conclusion, IFN-γ inhibits the expression of the integrin αvß3, reducing the cell adhesion to FN and the following intracellular signaling in thyroid cells in culture. These results suggest that integrins may be a target of the infiltrating lymphocytes and have a role in the pathogenesis of autoimmune thyroiditis.

EGFR is a transducer of the urokinase receptor initiated signal that is required for in vivo growth of a human carcinoma.

Urokinase plasminogen activator receptor (uPAR) activates alpha5beta1 integrin and ERK signaling, inducing in vivo proliferation of HEp3 human carcinoma. Here we demonstrate that EGFR mediates the uPAR/integrin/fibronectin (FN) induced growth pathway. Its activation is ligand-independent and does not require high EGFR, but does require high uPAR expression. Only when uPAR level is constitutively elevated does EGFR become alpha5beta1-associated and activated. Domain 1 of uPAR is crucial for EGFR activation, and FAK links integrin and EGFR signaling. Inhibition of EGFR kinase blocks uPAR induced signal to ERK, implicating EGFR as an important effector of the pathway. Disruption of uPAR or EGFR signaling reduces HEp3 proliferation in vivo. These findings unveil a mechanism whereby uPAR subverts ligand-regulated EGFR signaling, providing cancer cells with proliferative advantage.

Cell-Matrix interactions, the role of fibronectin and integrins. A survey.

In this review, we present several aspects of cell-matrix interactions, especially the role of fibronectin and integrins in the mediation of these interactions. As this field of investigations literally exploded over the last decades, we had to limit this review to some aspects of this field. We cited experiments giving details on the modifications of fibronectin molecules during their interactions with cells as well as on recent progress of the molecular mechanisms of fibronectin-integrin interactions. We insisted on the molecular details which were shown to play a role in the bi-directional signals "sent" by cells to the surrounding matrix (inside-out and outside-in). A number of recent publications confirmed the physiopathological importance of these messages both for the normal function of tissues as well as for the understanding of their pathological modifications. We insist also on the importance of fibronectin-fragments during some pathologies.

Genetic analyses of integrin function in mice.

Recent mutations of most integrin genes in the mouse have provided new exciting insights into the role of these integrins in cell-extracellular matrix interactions during development. The embryonic lethal phenotypes obtained by ablating integrins which are predominantly expressed in the mesenchyme confirmed the essential function of those integrins in morphogenesis. In contrast, null alleles for several epithelial integrins which bind components of basement membranes showed milder phenotypes, suggesting the presence of novel and unexpected redundant and compensatory mechanisms.

Selective regulation of integrin--cytoskeleton interactions by the tyrosine kinase Src.

Cell motility on extracellular-matrix (ECM) substrates depends on the regulated generation of force against the substrate through adhesion receptors known as integrins. Here we show that integrin-mediated traction forces can be selectively modulated by the tyrosine kinase Src. In Src-deficient fibroblasts, cell spreading on the ECM component vitronectin is inhibited, while the strengthening of linkages between integrin vitronectin receptors and the force-generating cytoskeleton in response to substrate rigidity is dramatically increased. In contrast, Src deficiency has no detectable effects on fibronectin-receptor function. Finally, truncated Src (lacking the kinase domain) co-localizes to focal-adhesion sites with alpha v but not with beta 1 integrins. These data are consistent with a selective, functional interaction between Src and the vitronectin receptor that acts at the integrin-cytoskeleton interface to regulate cell spreading and migration.

Stimulation of integrin-mediated adhesion of T lymphocytes and monocytes: two mechanisms with divergent biological consequences.

We show that the adhesion of T lymphoid cells to immobilized fibronectin can be increased by two distinct mechanisms. The first is by increasing the affinity of the fibronectin receptor/ligand interaction using the anti-beta 1 integrin monoclonal antibody 8A2. The second is by treating the cells with phorbol 12-myristate 13-acetate (PMA), which alters events that occur after receptor occupancy (e.g., cell spreading) without affecting receptor affinity. The effects of these two mechanisms on adhesion in the presence of physiological concentrations of soluble fibronectin suggest that they have different biological consequences. Under these conditions, the net effect of increasing the affinity of the fibronectin receptors is to decrease cell adhesion, whereas the increase in adhesion induced by PMA is unaffected. This suggests that the high affinity receptors are not primarily available for cell adhesion under these circumstances, and that they have an alternative function. We further show that high affinity binding of soluble fibronectin can be induced by either differentiation of the monocytic cell line THP-1 or by cross-linking the T cell receptor complexes on the T lymphoid cell line HUT-78. The differentiated monocytic cells express two populations of fibronectin receptors: a minority in a high affinity state, and the majority in a low affinity state. Thus they will both continue to adhere in the presence of physiological concentrations of soluble fibronectin and bind significant amounts of soluble fibronectin at the cell surface.

Caspase-8 is involved in neovascularization-promoting progenitor cell functions.

OBJECTIVE: Endothelial progenitor cells (EPCs) comprise a heterogeneous population of cells, which improve therapeutic neovascularization after ischemia. The neovascularization-promoting potential of progenitor cells depends on survival and retention of the infused cells to the tissue. Caspases mediate apoptosis but are also involved in other critical biological processes. Therefore, we aimed to address the role of caspases in proangiogenic cells. METHODS AND RESULTS: The caspase-8 inhibitor zIETD abrogated the ex vivo formation of EPCs, inhibited EPC adhesion and migration, and reduced their capacity to improve neovascularization in vivo. Consistently, cells isolated from caspase-8-deficient mice exhibited a reduced capacity for enhancing neovascularization when transplanted into mice after hindlimb ischemia. Because inhibition of Caspase-8 reduced the adhesion and homing functions of EPCs, we further determined the surface expression of integrins and receptors involved in cell recruitment to ischemic tissues. Pharmacological inhibition of caspase-8 and genetic depletion of caspase-8 reduced the expression of the fibronectin receptor subunits alpha5 and beta1 and the SDF-1 receptor CXCR4. Moreover, we identified the E3 ubiquitin ligase Cbl-b, which negatively regulates integrin and receptor-mediated signaling, as a potential Caspase-8 substrate. CONCLUSIONS: In summary, our data demonstrate a novel apoptosis-unrelated role of caspase-8 in proangiogenic cells.

Affinity modulation of integrin alpha 5 beta 1: regulation of the functional response by soluble fibronectin.

We report that a beta 1 integrin (alpha 5 beta 1) can exist in different affinity states for its soluble ligand, fibronectin. The alpha 5 beta 1 expressed by the erythroleukemic cell line K562 binds soluble fibronectin with low affinity (Kd > 1 microM), but is induced to bind it with 20-fold higher affinity (Kd-54 nM) in the presence of the anti-beta 1 mAb 8A2. This activation seems to be due to direct antibody-induced change in the receptor that does not require intracellular signaling, and is a plausible basis for the 8A2-induced enhancement of beta 1-dependent adhesion to fibronectin and other immobilized ligands (Kovach, N. L., T. M. Carlos, E. Yee, and J. M. Harlan. 1992. J. Cell Biol. 116: 499-509). Fab fragments of 8A2 bind with higher affinity to alpha 5 beta 1 receptor that is occupied by the GRG-DSP peptide ligand suggesting that the antibody functions by stabilizing a high affinity (occupied) conformer of the receptor. A functional consequence of the affinity modulation is that soluble fibronectin (at physiological concentrations) occupies the high affinity receptors, and so becomes an effective inhibitor of adhesion to immobilized fibronectin. In contrast, the majority of low affinity receptors remain unoccupied and are still to mediate cellular adhesion.

Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly.

The ability of single subunit chimeric receptors containing various integrin beta intracellular domains to mimic and/or inhibit endogenous integrin function was examined. Chimeric receptors consisting of the extracellular and transmembrane domains of the small subunit of the human interleukin-2 receptor connected to either the beta 1, beta 3, beta 3B, or beta 5 intracellular domain were transiently expressed in normal human fibroblasts. When expressed at relatively low levels, the beta 3 and beta 5 chimeras mimicked endogenous ligand-occupied integrins and, like the beta 1 chimera (LaFlamme, S. E., S. K. Akiyama, and K. M. Yamada. 1992. J. Cell Biol. 117:437), concentrated with endogenous integrins in focal adhesions and sites of fibronectin fibril formation. In contrast, the chimeric receptor containing the beta 3B intracellular domain (a beta 3 intracellular domain modified by alternative splicing) was expressed diffusely on the cell surface, indicating that alternative splicing can regulate integrin receptor distribution by an intracellular mechanism. Furthermore, when expressed at higher levels, the beta 1 and beta 3 chimeric receptors functioned as dominant negative mutants and inhibited endogenous integrin function in localization to fibronectin fibrils, fibronectin matrix assembly, cell spreading, and cell migration. The beta 5 chimera was a less effective inhibitor, and the beta 3B chimera and the reporter lacking an intracellular domain did not inhibit endogenous integrin function. Comparison of the relative levels of expression of the transfected beta 1 chimera and the endogenous beta 1 subunit indicated that in 10 to 15 h assays, the beta 1 chimera can inhibit cell spreading when expressed at levels approximately equal to the endogenous beta 1 subunit. Levels of chimeric receptor expression that inhibited cell spreading also inhibited cell migration, whereas lower levels were able to inhibit alpha 5 beta 1 localization to fibrils and matrix assembly. Our results indicate that single subunit chimeric integrins can mimic and/or inhibit endogenous integrin receptor function, presumably by interacting with cytoplasmic components critical for endogenous integrin function. Our results also demonstrate that beta intracellular domains, expressed in this context, display specificity in their abilities to mimic and inhibit endogenous integrin function. Furthermore, the approach that we have used permits the analysis of intracellular domain function in the processes of cell spreading, migration and extracellular matrix assembly independent of effects due to the rest of integrin dimers. This approach should prove valuable in the further analysis of integrin intracellular domain function in these and other integrin-mediated processes requiring the interaction of integrins with cytoplasmic components.

A point mutation of integrin beta 1 subunit blocks binding of alpha 5 beta 1 to fibronectin and invasin but not recruitment to adhesion plaques.

A point mutation in a highly conserved region of the beta 1 subunit, Asp130 to Ala (D130A) substitution, abrogates the Arg-Gly-Asp (RGD)-dependent binding of alpha 5 beta 1 to fibronectin (FN) without disrupting gross structure or heterodimer assembly. The D130A mutation also interferes with binding to invasin, a ligand that lacks RGD sequence. In spite of the lack of detectable FN binding by alpha 5 beta 1(D130A), it was recruited to adhesion plaques formed on FN by endogenous hamster receptors. Thus, intact ligand binding function is not required for recruitment of alpha 5 beta 1 to adhesion plaques. Overexpression of beta 1(D130A) partially interfered with endogenous alpha 5 beta 1 function, thus defining a dominant negative beta 1 integrin mutation.

A molecular mechanism of integrin crosstalk: alphavbeta3 suppression of calcium/calmodulin-dependent protein kinase II regulates alpha5beta1 function.

Many cells express more than one integrin receptor for extracellular matrix, and in vivo these receptors may be simultaneously engaged. Ligation of one integrin may influence the behavior of others on the cell, a phenomenon we have called integrin crosstalk. Ligation of the integrin alphavbeta3 inhibits both phagocytosis and migration mediated by alpha5beta1 on the same cell, and the beta3 cytoplasmic tail is necessary and sufficient for this regulation of alpha5beta1. Ligation of alpha5beta1 activates the calcium- and calmodulin-dependent protein kinase II (CamKII). This activation is required for alpha5beta1-mediated phagocytosis and migration. Simultaneous ligation of alphavbeta3 or expression of a chimeric molecule with a free beta3 cytoplasmic tail prevents alpha5beta1-mediated activation of CamKII. Expression of a constitutively active CamKII restores alpha5beta1 functions blocked by alphavbeta3-initiated integrin crosstalk. Thus, alphavbeta3 inhibition of alpha5beta1 activation of CamKII is required for its role in integrin crosstalk. Structure-function analysis of the beta3 cytoplasmic tail demonstrates a requirement for Ser752 in beta3-mediated suppression of CamKII activation, while crosstalk is independent of Tyr747 and Tyr759, implicating Ser752, but not beta3 tyrosine phosphorylation in initiation of the alphavbeta3 signal for integrin crosstalk.

Integrin activation by regulated dimerization and oligomerization of platelet endothelial cell adhesion molecule (PECAM)-1 from within the cell.

Platelet endothelial cell adhesion molecule (PECAM)-1 is a 130-kD transmembrane glycoprotein having six Ig homology domains within its extracellular domain and an immunoreceptor tyrosine-based inhibitory motif within its cytoplasmic domain. Previous studies have shown that addition of bivalent anti-PECAM-1 mAbs to the surface of T cells, natural killer cells, neutrophils, or platelets result in increased cell adhesion to immobilized integrin ligands. However, the mechanism by which this occurs is not clear, and it is possible that anti-PECAM-1 mAbs elicit this effect by simply sequestering PECAM-1, via antibody-induced patching and capping, away from stimulatory receptors that it normally regulates. To determine whether dimerization or oligomerization of PECAM-1 directly initiates signal transduction pathways that affect integrin function in an antibody-independent manner, stable human embryonic kidney-293 cell lines were produced that expressed chimeric PECAM-1 cDNAs containing one or two FK506-binding protein (FKBP) domains at their COOH terminus. Controlled dimerization initiated by addition of the bivalent, membrane-permeable FKBP dimerizer, AP1510, nearly doubled homophilic binding capacity, whereas AP1510-induced oligomers favored cis PECAM-1/PECAM-1 associations within the plane of the plasma membrane at the expense of trans homophilic adhesion. Importantly, AP1510-induced oligomerization resulted in a marked increase in both adherence and spreading of PECAM/FKBP-2-transfected cells on immobilized fibronectin, a reaction that was mediated by the integrin alpha(5)beta(1). These data demonstrate that signals required for integrin activation can be elicited by clustering of PECAM-1 from inside the cell, and suggest that a dynamic equilibrium between PECAM-1 monomers, dimers, and oligomers may control cellular activation signals that influence the adhesive properties of vascular cells that express this novel member of the immunoreceptor tyrosine-based inhibitory motif family of regulatory receptors.

The alpha v beta 1 integrin functions as a fibronectin receptor but does not support fibronectin matrix assembly and cell migration on fibronectin.

The fibronectin receptor, alpha 5 beta 1, has been shown to be required for fibronectin matrix assembly and plays an important role in cell migration on fibronectin. However, it is not clear whether other fibronectin binding integrins can take the place of alpha 5 beta 1 during matrix assembly and cell migration. To test this, we expressed the human alpha v subunit in the CHO cell line CHO-B2 that lacks the alpha 5 subunit. We found that the human alpha v combined with CHO cell beta 1 to form the integrin alpha v beta 1. Cells that expressed alpha v beta 1 attached to and spread well on fibronectin-coated dishes, but did so less well on vitronectin-coated dishes. This, along with other data, indicated that alpha v beta 1 functions as a fibronectin receptor in CHO-B2 cells. The alpha v beta 1-expressing cells failed to produce a fibronectin matrix or to migrate on fibronectin, although the same cells transfected with alpha 5 do produce a matrix and migrate on fibronectin. The affinity of the alpha v beta 1-expressing cells for fibronectin was fourfold lower than that of the alpha 5 beta 1-expressing cells. In addition, alpha v beta 1 was distributed diffusely throughout the cell surface, whereas alpha 5 beta 1 was localized to focal adhesions when cells were seeded onto fibronectin-coated surfaces. Thus, of the two fibronectin receptors, alpha v beta 1 and alpha 5 beta 1, only alpha 5 beta 1 supports fibronectin matrix assembly and promotes cell migration on fibronectin in the CHO-B2 cells. Possible reasons for this difference in the activities of alpha v beta 1 and alpha 5 beta 1 include the lower affinity of alpha v beta 1 for fibronectin and the failure of this integrin to localize in adhesion plaques on a fibronectin substrate. These results show that two integrins with similar ligand specificities and cell attachment functions may be quite different in their ability to support fibronectin matrix assembly and cell motility on fibronectin.

Integrin beta 3 cytoplasmic tail is necessary and sufficient for regulation of alpha 5 beta 1 phagocytosis by alpha v beta 3 and integrin-associated protein.

Using a K562 cell transfection model, we have previously described a novel relationship between the integrins alpha v beta 3 and alpha 5 beta 1. alpha v beta 3 ligation was able to inhibit alpha 5 beta 1-mediated phagocytosis without effect on alpha 5 beta 1-mediated adhesion. The alpha v beta 3-dependent inhibition apparently required a signal transduction cascade as it was reversed by inhibitors of serine/threonine kinases. Now, we have studied the mechanisms of signal transduction in this system and have found that the beta 3 cytoplasmic tail is both necessary and sufficient for initiation of the signal leading to inhibition of alpha 5 beta 1 phagocytosis. Ligation of integrin-associated protein (IAP), which has been implicated in alpha v beta 3 signal transduction, mimics the effects of alpha v beta 3 ligation only when the beta 3 integrin with an intact cytoplasmic tail is present. Although fibronectin-mediated phagocytosis requires the high affinity conformation of alpha 5 beta 1, ligation of alpha v beta 3/IAP does not prevent acquisition of this high affinity state. We conclude that alpha v beta 3/IAP ligation initates a signal transduction cascade, dependent upon the beta 3 cytoplasmic tail, which inhibits the phagocytic function of alpha 5 beta 1 at a step subsequent to modulation of integrin affinity.

The L1 adhesion molecule is a cellular ligand for VLA-5.

The L1 adhesion molecule is a member of the immunoglobulin superfamily shared by neural and immune cells. In the nervous system L1 can mediate cell binding by a homophilic mechanism. To analyze its function on leukocytes we studied whether L1 could interact with integrins. Here we demonstrate that VLA-5, an RGD-specific fibronectin receptor on a wide variety of cell types, can bind to murine L1. Mouse ESb-MP cells expressing VLA-5 and L1 could be induced to aggregate in the presence of specific mAbs to CD24 (heat-stable antigen), a highly and heterogeneously glycosylated glycophosphatidylinositol-linked differentiation antigen of hematopoietic and neural cells. The aggregation was blocked by both mAbs to L1 and VLA-5, respectively. Aggregation was blocked also by a synthetic RGD-containing peptide derived from the Ig-domain VI of the L1 protein. ESb-MP subclones with low L1 expression could not aggregate. In heterotypic binding assays mouse bone marrow cells could adhere in an L1-dependent fashion to platelets that expressed VLA-5. Also purified L1 coated to polystyrene beads could bind to platelets. The binding of L1-beads was again inhibited by mAbs to L1 and VLA-5, by soluble L1 and the L1-RGD peptide in a dose-dependent manner. Thymocytes or human Nalm-6 tumor cells expressing VLA-5 could adhere to affinity-purified L1 and to the L1-derived RGD-containing peptide coated to glass slides. The adhesion was strongly enhanced in the presence of Mn(2+)-ions and blocked by mAbs to VLA-5. We also demonstrate a direct L1-VLA-5 protein interaction. Our results suggest a novel binding pathway, in which the VLA-5 integrin binds to L1 on adjacent cells. Given its rapid downregulation on lymphocytes after induction of cell proliferation, L1 may be important in integrin-mediated and activation-regulated cell-cell interactions.

Defining fibronectin's cell adhesion synergy site by site-directed mutagenesis.

Fibronectin's RGD-mediated binding to the alpha5beta1 integrin is dramatically enhanced by a synergy site within fibronectin III domain 9 (FN9). Guided by the crystal structure of the cell-binding domain, we selected amino acids in FN9 that project in the same direction as the RGD, presumably toward the integrin, and mutated them to alanine. R1379 in the peptide PHSRN, and the nearby R1374 have been shown previously to be important for alpha5beta1-mediated adhesion (Aota, S., M. Nomizu, and K.M. Yamada. 1994. J. Biol. Chem. 269:24756-24761). Our more extensive set of mutants showed that R1379 is the key residue in the synergistic effect, but other residues contribute substantially. R1374A decreased adhesion slightly by itself, but the double mutant R1374A-R1379A was significantly less adhesive than R1379A alone. Single mutations of R1369A, R1371A, T1385A, and N1386A had negligible effects on cell adhesion, but combining these substitutions either with R1379A or each other gave a more dramatic reduction of cell adhesion. The triple mutant R1374A/P1376A/R1379A had no detectable adhesion activity. We conclude that, in addition to the R of the PHRSN peptide, other residues on the same face of FN9 are required for the full synergistic effect. The integrin-binding synergy site is a much more extensive surface than the small linear peptide sequence.