Cross-talk between insulin receptor and integrin alpha5 beta1 signaling pathways

The ligation and clustering of cell surface alphabeta heterodimeric integrins enhances cell adhesion and initiates signaling pathways that regulate such processes as cell spreading, migration, differentiation, proliferation and apoptosis. Here we show that insulin treatment of Chinese hamster ovary cells expressing insulin receptors (CHO-T) markedly promotes cell adhesion onto a fibronectin matrix, but not onto bovine serum albumin or poly-lysine. Incubation of cells with a GRGDSP peptide that specifically binds integrins (but not the nonspecific GRADSP peptide) abolishes this insulin effect, as does the potent phosphoinositide 3-kinase (PI 3-kinase) inhibitor wortmannin. Moreover, a specific blocking monoclonal anti-alpha5beta1 integrin antibody, PB-1, blocks insulin-stimulated cell adhesion onto fibronectin. Conversely, activating alpha5beta1 integrins on CHO-T cells by adherence onto fibronectin markedly potentiates the action of insulin to enhance insulin receptor and insulin receptor substrate (IRS)-1 tyrosine phosphorylation. Activation of alpha5beta1 integrin also markedly potentiates the recruitment of p85-associated PI 3-kinase activity to IRS-1 in response to submaximal levels of insulin in CHO-T cells. These data indicate that insulin potently activates integrin alpha5beta1 mediated CHO-T cell adhesion, while integrin alpha5beta1 signaling in turn enhances insulin receptor kinase activity and formation of complexes containing IRS-1 and PI 3-kinase. These findings raise the hypothesis that insulin receptor and alpha5beta1 integrin signaling act synergistically to enhance cell adhesion.     

A functional role for specific spliced variants of the alpha7beta1 integrin in acetylcholine receptor clustering

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the alpha7beta1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, alpha7A and alpha7B, and the extracellular spliced forms, alpha7X1 and alpha7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the alpha7beta1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-alpha7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-alpha7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the alpha7A and alpha7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the alpha7X2 extracellular domain were active. These results demonstrate that the alpha7beta1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the alpha7 chain, and that laminin, agrin, and the alpha7beta1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.     

Low density lipoprotein phosphorylates the focal adhesion-associated kinase p125(FAK) in human platelets independent of integrin alphaIIb beta3

Low density lipoprotein (LDL) is known to sensitize platelets to agonists via integrin mediated outside-in signaling (Hackeng, C. M., Huigsloot, M., Pladet, M. W., Nieuwenhuis, H. K., Rijn, H. J. M. v., and Akkerman, J. W. N. (1999) Arterioscler. Thromb. Vasc. Biol., in press). As outside in signaling is associated with phosphorylation of p125(FAK), the effect of LDL on p125(FAK) phosphorylation in platelets was investigated. LDL induced p125(FAK) phosphorylation in a dose- and time- dependent manner. The phosphorylation was independent of ligand binding to integrin alphaIIbbeta3 and aggregation, such in contrast to alpha-thrombin-induced p125(FAK) phosphorylation, that critically depended on platelet aggregation. Platelets from patients with Glanzmann's thrombastenia showed the same LDL- induced phos- phorylation of p125(FAK) as control platelets, whereas alpha-thrombin completely failed to phosphorylate the kinase in the patients platelets. LDL signaling to p125(FAK) was independent of integrin alpha2 beta1, the FcgammaRII receptor, and the lysophosphatidic acid receptor and not affected by inhibitors of cyclooxygenase, protein kinase C, ERK1/2 or p38(MAPK). Phosphorylation of p125(FAK) by LDL was strongly inhibited by cyclic AMP. These observations indicate that LDL is a unique platelet agonist, as it phosphorylates p125(FAK) in platelet suspensions, under unstirred conditions and independent of integrin alphaIIb beta3.     

Regulation of alphaIIb beta3 function in human B lymphocytes

We studied the function of the platelet integrin alphaIIb beta3 using a B lymphocyte model in which alphaIIb beta3 can be induced to interact with fibrinogen using phorbol myristate acetate (PMA). To determine whether a G protein-coupled receptor could also activate alphaIIb beta3 in lymphocytes, we coexpressed the human formyl peptide receptor (fPR) and alphaIIb beta3, finding that the fPR agonist formyl Met-Leu-Phe (fMLP)-stimulated lymphocyte adherence to immobilized fibrinogen and binding of soluble fibrinogen to the lymphocyte surface. The response to fMLP, but not PMA, was abrogated by pertussis toxin, indicating that the fPR was coupled to the G-protein Galphai, whereas the protein kinase C inhibitor bisindolylmaleimide I inhibited the response to both fMLP and PMA, indicating that signaling from the fPR included protein kinase C. On the other hand, the tyrosine kinase inhibitor genistein, the Syk inhibitor piceatannol, and the RhoA inhibitor C3 exoenzyme had no effect, implying that neither tyrosine phosphorylation nor the GTPase RhoA were involved. Furthermore, whereas micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of alphaIIb beta3 with fibrinogen, nanomolar concentrations actually induced fibrinogen binding to unstimulated cells. Our studies demonstrate that alphaIIb beta3 expressed in B lymphocytes can be activated by a physiologic agonist and outline an activating pathway that includes Galphai, protein kinase C, and the actin cytoskeleton.     

Association between ligand-induced conformational changes of integrin IIbbeta3 and IIbbeta3-mediated intracellular Ca2+ signaling

Platelet IIbbeta3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of IIbbeta3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and IIbbeta3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various IIbbeta3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as IIbbeta3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both IIb and beta3 subunits. In group II, antagonists can induce LIBS on the IIb subunit, but not on the beta3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on IIbbeta3. Neither group I nor group II antagonist increased intracellular Ca2+ concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to IIbbeta3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable IIbbeta3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate IIbbeta3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between beta3 LIBS expression and IIbbeta3-mediated intracellular Ca2+ signaling in platelets.     

Examination of ligand-induced conformational changes in the beta2 adrenergic receptor

The environmentally sensitive and cysteine reactive fluorescent probe, IANBD, was used to monitor ligand-induced structural changes in the beta2 adrenergic receptor (beta2AR) by fluorescent spectroscopy. We found that agonists caused a dose-dependent and reversible decrease in fluorescence from the purified IANBD-labeled beta2AR. This suggested that agonists promote a conformational change in the receptor that leads to an increase in the polarity of the environment around one or more IANBD labeled cysteines. The wildtype receptor contains eight free cysteines and mutagenesis and peptide mapping experiments have indicated that several of these sites are accessible for chemical derivatization. Thus, to identify the cysteine(s) involved in the agonist-induced change in fluorescence and thereby map agonist-induced conformational changes in the beta2AR, we generated a series of mutant receptors having limited numbers of cysteines available for fluorescent labeling. Fluorescence spectroscopy analysis of the purified and site-selectively IANBD-labeled mutants showed that IANBD labeled 125Cys and 285Cys are responsible for the observed changes in fluorescence consistent with movements of TM III and VI in response to agonist binding.     

Requirement of vascular integrin alpha v beta 3 for angiogenesis

Angiogenesis depends on the adhesive interactions of vascular cells. The adhesion receptor integrin alpha v beta 3 was identified as a marker of angiogenic vascular tissue. Integrin alpha v beta 3 was expressed on blood vessels in human wound granulation tissue but not in normal skin, and it showed a fourfold increase in expression during angiogenesis on the chick chorioallantoic membrane. In the latter assay, a monoclonal antibody to alpha v beta 3 blocked angiogenesis induced by basic fibroblast growth factor, tumor necrosis factor-alpha, and human melanoma fragments but had no effect on preexisting vessels. These findings suggest that alpha v beta 3 may be a useful therapeutic target for diseases characterized by neovascularization.     

Distinct involvement of beta3 integrin cytoplasmic domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal assembly and phosphotyrosine signaling

We have investigated the structural requirements of the beta3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during alphav beta3-mediated cell spreading. Using CHO cells transfected with various beta3 mutants, we demonstrate a close correlation between alphav beta3-mediated cell spreading and tyrosine phosphorylation of FAK and paxillin, and highlight a distinct involvement of the NPLY747 and NITY759 motifs in these signaling processes. Deletion of the NITY759 motif alone was sufficient to completely prevent alphav beta3-dependent focal contact formation, cell spreading, and FAK/paxillin phosphorylation. The single Y759A substitution induced a strong inhibitory phenotype, while the more conservative, but still phosphorylation-defective, Y759F mutation restored wild type receptor function. Alanine substitution of the highly conserved Tyr747 completely abolished alphav beta3-dependent formation of focal adhesion plaques, cell spreading, and FAK/paxillin phosphorylation, whereas a Y747F substitution only partially restored these events. As none of these mutations affected receptor-ligand interaction, our results suggest that the structural integrity of the NITY759 motif, rather than the phosphorylation status of Tyr759 is important for beta3-mediated cytoskeleton reorganization and tyrosine phosphorylation of FAK and paxillin, while the presence of Tyr at residue 747 within the NPLY747 motif is required for optimal beta3 post-ligand binding events.     

Genetic analysis of the Drosophila alphaPS2 integrin subunit reveals discrete adhesive, morphogenetic and sarcomeric functions

The integrin family of cell surface receptors mediates cell-substrate and cell-to-cell adhesion and transmits intracellular signals. In Drosophila there is good evidence for an adhesive role of integrins, but evidence for integrin signalling has remained elusive. Each integrin is an alphabeta heterodimer, and the Drosophila betaPS subunit forms at least two integrins by association with different alpha subunits: alphaPS1betaPS (PS1) and alphaPS2betaPS (PS2). The complex pattern of PS2 integrin expression includes, but is more extensive than, the sites where PS2 has a known requirement. In order to investigate whether PS2 integrin is required at these additional sites and/or has functions besides mediating adhesion, a comprehensive genetic analysis of inflated, the gene that encodes alphaPS2, was performed. We isolated 35 new inflated alleles, and obtained 10 alleles from our colleagues. The majority of alleles are amorphs (36/45) or hypomorphs (4/45), but five alleles that affect specific developmental processes were identified. Interallelic complementation between these alleles suggests that some may affect distinct functional domains of the alphaPS2 protein, which specify particular interactions that promote adhesion or signalling. One new allele reveals that the PS2 integrin is required for the development of the adult halteres and legs as well as the wing.     

Phosphorylation sites in the integrin beta3 cytoplasmic domain in intact platelets

Protein seryl/threonyl phosphatase inhibitors such as calyculin A block inside-out and outside-in platelet signaling. Our studies demonstrate that the addition of calyculin A blocks platelet adhesion and spreading on fibrinogen, responses that depend on integrin alphaIIb beta3 signaling. We hypothesized that this reflects a change in alphaIIb beta3 structure caused by a specific state of phosphorylation. We show that addition of calyculin A leads to increased phosphorylation of the beta3 subunit, and phosphoamino acid analysis reveals that only threonine residues become phosphorylated; sequence analysis by Edman degradation established that threonine 753 became stoichiometrically phosphorylated during inhibition of platelet phosphatases by calyculin A. This region of beta3 is linked to outside-in signaling such as platelet spreading responses. The effect of calyculin A on platelet adhesion and spreading and on the phosphorylation of T-753 in beta3 is reversed by the calcium ionophore A23187, demonstrating that these effects of calyculin A are not generally toxic ones. We propose that phosphorylation of beta3 on threonine 753, a region of beta3 linked to outside-in signaling, may be a mechanism by which integrin alphaIIb beta3 function is regulated.     

A conserved sequence motif in the integrin beta3 cytoplasmic domain is required for its specific interaction with beta3-endonexin

Integrin signaling is mediated by interaction of integrin cytoplasmic domains with intracellular signaling molecules. Recently, we identified a novel 111-amino acid polypeptide, termed beta3-endonexin, which interacts selectively with the integrin beta3 cytoplasmic domain. In the present study we conducted a systematic mutational analysis of both the integrin beta3 cytoplasmic domain and beta3-endonexin to map sites required for interaction. The interaction of the full-length beta3 integrin subunit with beta3-endonexin in vitro required the beta3 cytoplasmic domain. In a yeast two-hybrid system, both membrane-proximal and membrane-distal residues of the beta3 cytoplasmic domain were necessary for interaction with beta3-endonexin. In particular, the membrane-distal NITY motif at beta3 756-759 was critical for the interaction. Exchange of beta3 residues 756-759 (NITY) for the corresponding residues in beta1 (NPKY) endowed the beta1 cytoplasmic domain with the ability to interact with beta3-endonexin. Conversely, exchange of the NPKY motif at beta1 772-775 for the NITY motif in beta3 abolished interaction of this chimeric cytoplasmic domain with beta3-endonexin. Because the NITY motif is present in the beta3 but not the beta1 cytoplasmic domain, these results explain the selective interaction of this cytoplasmic domain with beta3-endonexin. In addition, deletional analysis suggested that a core 91-residue sequence of beta3-endonexin is sufficient for specific binding to the beta3 cytoplasmic domain. These studies have identified a cytoplasmic domain sequence motif that specifies an integrin-specific protein-protein interaction.     

Expression of fibronectin and its integrin receptor alpha 5 beta 1 in canine mammary tumours

Fibronectin and its integrin receptor alpha 5 beta 1 were studied by immunohistochemical methods in five normal canine mammary glands, four dysplastic glands and 18 mammary tumours. The aim of the study was to evaluate the possible changes in the alpha 5 beta 1 integrin receptor and its ligand fibronectin in relation to the metastatic capacity of canine mammary neoplasms. The immunostaining of alpha 5 beta 1 was very uniform in the hyperplastic glands but uneven in the mammary tumours. The expression of alpha 5 and beta 1 was diminished in metastatic tumours but there were some alpha 5-positive cells with pronounced features of malignancy and immaturity. Stromal fibronectin was increased in most cases and cytoplasmic staining of fibronectin was observed in epithelial and myoepithelial cells in mammary neoplasms but not in normal or dysplastic mammary tissue. There was no relationship between the content of alpha 5 beta 1 and the expression of fibronectin in canine mammary tumours.     

Identification of osteopontin as a novel ligand for the integrin alpha8 beta1 and potential roles for this integrin-ligand interaction in kidney morphogenesis

Epithelio-mesenchymal interactions during kidney organogenesis are disrupted in integrin alpha8 beta1-deficient mice. However, the known ligands for integrin alpha8 beta1-fibronectin, vitronectin, and tenascin-C-are not appropriately localized to mediate all alpha8 beta1 functions in the kidney. Using a method of general utility for determining the distribution of unknown integrin ligands in situ and biochemical characterization of these ligands, we identified osteopontin (OPN) as a ligand for alpha8 beta1. We have coexpressed the extracellular domains of the mouse alpha8 and beta1 integrin subunits as a soluble heterodimer with one subunit fused to alkaline phosphatase (AP) and have used the alpha8 beta1-AP chimera as a histochemical reagent on sections of mouse embryos. Ligand localization with alpha8 beta1-AP in developing bone and kidney was observed to be overlapping with the distribution of OPN. In "far Western" blots of mouse embryonic protein extracts, bands were detected with sizes corresponding to fibronectin, vitronectin, and unknown proteins, one of which was identical to the size of OPN. In a solid-phase binding assay we demonstrated that purified OPN binds specifically to alpha8 beta1-AP. Cell adhesion assays using K562 cells expressing alpha8 beta1 were used to confirm this result. Together with a recent report that anti-OPN antibodies disrupt kidney morphogenesis, our results suggest that interactions between OPN and integrin alpha8 beta1 may help regulate kidney development and other morphogenetic processes.     

Interleukin 2-mediated uncoupling of T cell receptor alpha/beta from CD3 signaling

T cell activation and clonal expansion is the result of the coordinated functions of the receptors for antigen and interleukin (IL)-2. The protein tyrosine kinase p56(lck) is critical for the generation of signals emanating from the T cell antigen receptor (TCR) and has also been demonstrated to play a role in IL-2 receptor signaling. We demonstrate that an IL-2-dependent, antigen-specific CD4(+) T cell clone is not responsive to anti-TCR induced growth when propagated in IL-2, but remains responsive to both antigen and CD3epsilon-specific monoclonal antibody. Survival of this IL-2-dependent clone in the absence of IL-2 was supported by overexpression of exogenous Bcl-xL. Culture of this clonal variant in the absence of IL-2 rendered it susceptible to anti-TCR-induced signaling, and correlated with the presence of kinase-active Lck associated with the plasma membrane. The same phenotype is observed in primary, resting CD4(+) T cells. Furthermore, the presence of kinase active Lck associated with the plasma membrane correlates with the presence of ZAP 70-pp21zeta complexes in both primary T cells and T cell clones in circumstances of responsive anti-TCR signaling. The results presented demonstrate that IL-2 signal transduction results in the functional uncoupling of the TCR complex through altering the subcellular distribution of kinase-active Lck.     

Potential roles of osteopontin and alphaVbeta3 integrin in the development of coronary artery restenosis after angioplasty

Angioplasty procedures are increasingly used to reestablish blood flow in blocked atherosclerotic coronary arteries. A serious complication of these procedures is reocclusion (restenosis), which occurs in 30-50% of patients. Migration of coronary artery smooth muscle cells (CASMCs) to the site of injury caused by angioplasty and subsequent proliferation are suggested mechanisms of reocclusion. Using both cultured human CASMCs and coronary atherectomy tissues, we studied the roles of osteopontin (OPN) and one of its receptors, alphavbeta3 integrin, in the pathogenesis of coronary restenosis. We also measured the plasma levels of OPN before and after angioplasty and determined the effect of exogenous OPN on CASMC migration, extracellular matrix invasion, and proliferation. We found that cultured CASMCs during log phase of growth and smooth muscle cell layer of the coronary atherosclerotic tissues of patients express both OPN mRNA and protein at a significantly elevated level compared with controls. Interestingly, whereas the baseline plasma OPN levels in control samples were virtually undetectable, those in patient plasma were remarkably high. We also found that interaction of OPN with alphavbeta3 integrin, expressed on CASMCs, causes migration, extracellular matrix invasion, and proliferation. These effects were abolished when OPN or alphavbeta3 integrin gene expression in CASMCs was inhibited by specific antisense S-oligonucleotide treatment or OPN-alphavbeta3 interaction was blocked by treatment of CASMCs with antibodies against OPN or alphavbeta3 integrin. Our results demonstrate that OPN and alphavbeta3 integrin play critical roles in regulating cellular functions deemed essential for restenosis. In addition, these results raise the possibility that transient inhibition of OPN gene expression or blocking of OPN-alphavbeta3 interaction may provide a therapeutic approach to preventing restenosis.