Detection of integrin alpha IIbbeta 3 clustering in living cells

In platelets, bidirectional signaling across integrin alpha(IIb)beta(3) regulates fibrinogen binding, cytoskeletal reorganization, cell aggregation, and spreading. Because these responses may be influenced by the clustering of alpha(IIb)beta(3) heterodimers into larger oligomers, we established two independent methods to detect integrin clustering and evaluate factors that regulate this process. In the first, weakly complementing beta-galactosidase mutants were fused to the C terminus of individual alpha(IIb) subunits, and the chimeras were stably expressed with beta(3) in Chinese hamster ovary cells. Clustering of alpha(IIb)beta(3) should bring the mutants into proximity and reconstitute beta-galactosidase activity. In the second method, alpha(IIb) was fused to either a green fluorescent protein (GFP) or Renilla luciferase and transiently expressed with beta(3). Here, integrin clustering should stimulate bioluminescence resonance energy transfer between a cell-permeable luciferase substrate and GFP. These methods successfully detected integrin clustering induced by anti-alpha(IIb)beta(3) antibodies. Significantly, they also detected clustering upon soluble fibrinogen binding to alpha(IIb)beta(3). In contrast, no clustering was observed following direct activation of alpha(IIb)beta(3) by MnCl(2) or an anti-alpha(IIb)beta(3)-activating antibody Fab in the absence of fibrinogen. Intracellular events also influenced alpha(IIb)beta(3) clustering. For example, a cell-permeable, bivalent FK506-binding protein (FKBP) ligand stimulated clustering when added to cells expressing an alpha(IIb)(FKBP)(2) chimera complexed with beta(3). Furthermore, alpha(IIb)beta(3) clustering occurred in the presence of latrunculin A or cytochalasin D, inhibitors of actin polymerization. These effects were enhanced by fibrinogen, suggesting that actin-regulated clustering modulates alpha(IIb)beta(3) interaction with ligands. These studies in living cells establish that alpha(IIb)beta(3) clustering is modulated by fibrinogen and actin dynamics. More broadly, they should facilitate investigations of the mechanisms and consequences of integrin clustering.     

Calcium integrin-binding protein activates platelet integrin alpha IIbbeta 3.

alpha(IIb)beta(3), a platelet-specific integrin, plays a critical role in platelet aggregation. The affinity of alpha(IIb)beta(3) for its ligands such as fibrinogen and von Willebrand factor is tightly regulated in an uncharacterized intracellular process termed inside-out signaling. Calcium integrin-binding protein (CIB) has been identified as a protein interacting with the cytoplasmic tail of the alpha(IIb) subunit of alpha(IIb)beta(3), but its physiological role has not been defined. In the present study, I demonstrate that CIB activates alpha(IIb)beta(3) both in vitro and in vivo. CIB interacts directly with the alpha(IIb) cytoplasmic tail, thereby increasing the affinity of alpha(IIb)beta(3) for fibrinogen in an in vitro fibrinogen-binding assay. The interaction of CIB with the alpha(IIb) cytoplasmic tail is enhanced in a Ca(2+)-dependent manner. A physiological agonist, ADP, stimulates platelets, activating alpha(IIb)beta(3). When the interaction of CIB with the alpha(IIb) cytoplasmic tail is blocked in native platelets by a permeable competing peptide, alpha(IIb)beta(3) activation is not detected even in the presence of ADP. This result indicates that direct interaction of CIB with the alpha(IIb) cytoplasmic tail converts alpha(IIb)beta(3) from a resting to an active conformation. This suggests that CIB plays an important role in one of the pathways that modulate the affinity of alpha(IIb)beta(3) for its ligand.     

Distinct glycoprotein Ib/V/IX and integrin alpha IIbbeta 3-dependent calcium signals cooperatively regulate platelet adhesion under flow.

We have investigated the calcium signaling relationship between the two major platelet adhesion receptors, glycoprotein Ib/V/IX (GPIb/V/IX) and integrin alpha(IIb)beta(3), involved in regulating platelet adhesion on von Willebrand factor (vWf) under flow. Our studies demonstrate that GPIb engagement of immobilized vWf elicits a transient calcium spike that may function to promote reversible arrest of translocating platelets. Subsequent integrin alpha(IIb)beta(3) engagement of vWf promotes sustained calcium oscillations that are essential for the maintenance of irreversible adhesion. GPIb-induced calcium spikes appear distinct from those initiated by integrin alpha(IIb)beta(3), in that the former are exclusively mediated through release of intracellular calcium stores via a signaling mechanism independent of PI 3-kinase. In contrast, integrin alpha(IIb)beta(3)-dependent calcium flux involves a PI 3-kinase-dependent signaling mechanism linked to intracellular calcium mobilization and subsequent transmembrane calcium influx. Studies employing the caged calcium chelator (o-nitrophenyl-EGTA) demonstrate that transient calcium spikes initiate a transient phase of platelet arrest that is converted to irreversible adhesion with the development of sustained oscillatory calcium flux. These studies demonstrate the existence of a dual step calcium signaling mechanism utilized by GPIb and integrin alpha(IIb)beta(3) that serves to regulate the dynamics of platelet adhesion under flow.     

The platelet integrin alpha IIbbeta 3 has an endogenous thiol isomerase activity

Integrins are cysteine-rich heterodimeric cell-surface adhesion molecules that alter their affinity for ligands in response to cellular activation. The molecular mechanisms involved in this activation of integrins are not understood. Treatment with the thiol-reducing agent, dithiothreitol, can induce an activation-like state in many integrins suggesting that cysteine-cysteine dithiol bonds are important for the receptor's tertiary structure and may be involved in activation-induced conformational changes. Here we demonstrate that the platelet-specific integrin, alpha(IIb)beta(3), contains an endogenous thiol isomerase activity, predicted from the presence of the tetrapeptide motif, CXXC, in each of the cysteine-rich repeats of the beta(3) polypeptide. This motif comprises the active site in enzymes involved in disulfide exchange reactions, including protein-disulfide isomerase (EC ) and thioredoxin. Intrinsic thiol isomerase activity is also observed in the related integrin, alpha(v)beta(3), which shares a common beta-subunit. Thiol isomerase activity within alpha(IIb)beta(3) is time-dependent and saturable, and is inhibited by the protein-disulfide isomerase inhibitor, bacitracin. Furthermore, this activity is calcium-sensitive and is regulated in the EDTA-stabilized conformation of the integrin. This novel demonstration of an enzymatic activity associated with an integrin subunit suggests that altered thiol bonding within the integrin or its substrates may be locally modified during alpha(IIb)beta(3) activation.     

Red cell ICAM-4 is a novel ligand for platelet-activated alpha IIbbeta 3 integrin

ICAM-4 (LW blood group glycoprotein) is an erythroid-specific membrane component that belongs to the family of intercellular adhesion molecules and interacts in vitro with different members of the integrin family, suggesting a potential role in adhesion or cell interaction events, including hemostasis and thrombosis. To evaluate the capacity of ICAM-4 to interact with platelets, we have immobilized red blood cells (RBCs), platelets, and ICAM-Fc fusion proteins to a plastic surface and analyzed their interaction in cell adhesion assays with RBCs and platelets from normal individuals and patients, as well as with cell transfectants expressing the alpha(IIb)beta(3) integrin. The platelet fibrinogen receptor alpha(IIb)beta(3) (platelet GPIIb-IIIa) in a high affinity state following GRGDSP peptide activation was identified for the first time as the receptor for RBC ICAM-4. The specificity of the interaction was demonstrated by showing that: (i) activated platelets adhered less efficiently to immobilized ICAM-4-negative than to ICAM-4-positive RBCs, (ii) monoclonal antibodies specific for the beta(3)-chain alone and for a complex-specific epitope of the alpha(IIb)beta(3) integrin, and specific for ICAM-4 to a lesser extent, inhibited platelet adhesion, whereas monoclonal antibodies to GPIb, CD36, and CD47 did not, (iii) activated platelets from two unrelated type-I glanzmann's thrombasthenia patients did not bind to coated ICAM-4. Further support to RBC-platelet interaction was provided by showing that dithiothreitol-activated alpha(IIb)beta(3)-Chinese hamster ovary transfectants strongly adhere to coated ICAM-4-Fc protein but not to ICAM-1-Fc and was inhibitable by specific antibodies. Deletion of individual Ig domains of ICAM-4 and inhibition by synthetic peptides showed that the alpha(IIb)beta(3) integrin binding site encompassed the first and second Ig domains and that the G65-V74 sequence of domain D1 might play a role in this interaction. Although normal RBCs are considered passively entrapped in fibrin polymers during thrombus, these studies identify ICAM-4 as the first RBC protein ligand of platelets that may have relevant physiological significance.     

A mitogen-activated protein kinase-dependent signaling pathway in the activation of platelet integrin alpha IIbbeta3

We have recently shown that the platelet integrin alpha(IIb)beta(3) is activated by von Willebrand factor (vWF) binding to its platelet receptor, glycoprotein Ib-IX (GPIb-IX), via the protein kinase G (PKG) signaling pathway. Here we show that GPIb-IX-mediated activation of integrin alpha(IIb)beta(3) is inhibited by dominant negative mutants of Raf-1 and MEK1 in a reconstituted integrin activation model in Chinese hamster ovary (CHO) cells and that the integrin-dependent platelet aggregation induced by either vWF or low dose thrombin is inhibited by MEK inhibitors PD98059 and U0126. Thus, mitogen-activated protein kinase (MAPK) pathway is important in GPIb-IX-dependent activation of platelet integrin alpha(IIb)beta(3). Furthermore, vWF binding to GPIb-IX induces phosphorylation of Thr-202/Tyr-204 of extracellular signal-regulated kinase 2 (ERK2). GPIb-IX-induced ERK2 phosphorylation is inhibited by PKG inhibitors and enhanced by overexpression of recombinant PKG. PKG activators also induce ERK phosphorylation, indicating that activation of MAPK pathway is downstream from PKG. Thus, our data delineate a novel integrin activation pathway in which ligand binding to GPIb-IX activates PKG that stimulates MAPK pathway, leading to integrin activation.     

Relationships between Rap1b, affinity modulation of integrin alpha IIbbeta 3, and the actin cytoskeleton

The affinity of integrin alpha(IIb)beta(3) for fibrinogen is controlled by inside-out signals that are triggered by agonists like thrombin. Agonist treatment of platelets also activates Rap1b, a small GTPase known to promote integrin-dependent adhesion of other cells. Therefore, we investigated the role of Rap1b in alpha(IIb)beta(3) function by viral transduction of GFP-Rap1 chimeras into murine megakaryocytes, which exhibit inside-out signaling similar to platelets. Expression of constitutively active GFP-Rap1b (V12) had no effect on unstimulated megakaryocytes, but it greatly augmented fibrinogen binding to alpha(IIb)beta(3) induced by a PAR4 thrombin receptor agonist (p < 0.01). The Rap1b effect was cell-autonomous and was prevented by pre-treating cells with cytochalasin D or latrunculin A to inhibit actin polymerization. Rap1b-dependent fibrinogen binding to megakaryocytes was blocked by POW-2, a novel monovalent antibody Fab fragment specific for high affinity murine alpha(IIb)beta(3). In contrast to GFP-Rap1b (V12), expression of GFP-Rap1GAP, which deactivates endogenous Rap1, inhibited agonist-induced fibrinogen binding (p < 0.01), as did dominant-negative GFP-Rap1b (N17) (p < 0.05). None of these treatments affected surface expression of alpha(IIb)beta(3). These studies establish that Rap1b can augment agonist-induced ligand binding to alpha(IIb)beta(3) through effects on integrin affinity, possibly by modulating alpha(IIb)beta(3) interactions with the actin cytoskeleton.     

DOCK2 is required for chemokine-promoted human T lymphocyte adhesion under shear stress mediated by the integrin alpha4beta1

The alpha4beta1 integrin is an essential adhesion molecule for recruitment of circulating lymphocytes into lymphoid organs and peripheral sites of inflammation. Chemokines stimulate alpha4beta1 adhesive activity allowing lymphocyte arrest on endothelium and subsequent diapedesis. Activation of the GTPase Rac by the guanine-nucleotide exchange factor Vav1 promoted by CXCL12 controls T lymphocyte adhesion mediated by alpha4beta1. In this study, we investigated the role of DOCK2, a lymphocyte guanine-nucleotide exchange factor also involved in Rac activation, in CXCL12-stimulated human T lymphocyte adhesion mediated by alpha4beta1. Using T cells transfected with DOCK2 mutant forms defective in Rac activation or with DOCK2 small interfering RNA, we demonstrate that DOCK2 is needed for efficient chemokine-stimulated lymphocyte attachment to VCAM-1 under shear stress. Flow chamber, soluble binding, and cell spreading assays identified the strengthening of alpha4beta1-VCAM-1 interaction, involving high affinity alpha4beta1 conformations, as the adhesion step mainly controlled by DOCK2 activity. The comparison of DOCK2 and Vav1 involvement in CXCL12-promoted Rac activation and alpha4beta1-dependent human T cell adhesion indicated a more prominent role of Vav1 than DOCK2. These results suggest that DOCK2-mediated signaling regulates chemokine-stimulated human T lymphocyte alpha4beta1 adhesive activity, and that cooperation with Vav1 might be required to induce sufficient Rac activation for efficient adhesion. In contrast, flow chamber experiments using lymph node and spleen T cells from DOCK2(-/-) mice revealed no significant alterations in CXCL12-promoted adhesion mediated by alpha4beta1, indicating that DOCK2 activity is dispensable for triggering of this adhesion in mouse T cells, and suggesting that Rac activation plays minor roles in this process.     

Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets

Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. We show here that fibrillar collagen, which initiates platelet activation at the damaged vessel wall, activates only a small fraction of platelets in suspension directly, whereas the majority of platelets becomes activated by mediators released from collagen-activated platelets. In Galpha(q)-deficient platelets that do not respond with activation of integrin alpha(IIb)beta(3) to a variety of mediators like thromboxane A2 (TXA2), thrombin, or ADP, collagen at high concentrations was able to induce aggregation, an effect that could be blocked by antagonists of the TXA2 or P2Y12 receptors. The activation of TXA2 or P2Y12 receptors alone, which in Galpha(q)-deficient platelets couple to G12/G13 and Gi, respectively, did not induce platelet integrin activation or aggregation. However, concomitant activation of both receptors resulted in irreversible integrin alpha(IIb)beta3-mediated aggregation of Galpha(q)-deficient platelets. Thus, the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin alpha(IIb)beta3 activation. Although G(q)-mediated signaling plays an important role in platelet activation, it is not strictly required for the activation of integrin alpha(IIb)beta3. This indicates that the efficient induction of platelet aggregation through G-protein-coupled receptors is an integrated response mediated by various converging G-protein-mediated signaling pathways involving G(q) and G(i) as well as G12/G13.     

Lateral clustering of platelet GP Ib-IX complexes leads to up-regulation of the adhesive function of integrin alpha IIbbeta 3.

Binding of von Willebrand factor (VWF) to GP Ib-IX mediates initial platelet adhesion and increases the subsequent adhesive function of alpha(IIb)beta(3). Because these responses are promoted most effectively by large VWF multimers, we hypothesized that receptor clustering modulates GP Ib-IX function. To test this, GP IX was fused at its cytoplasmic tail to tandem repeats of FKBP, and GP Ib-IX(FKBP)(2) and alpha(IIb)beta(3) were expressed in Chinese hamster ovary cells. Under flow conditions at wall shear rates of up to 2000 s(-1), GP Ib-IX(FKBP)(2) mediated cell tethering to immobilized VWF, just as in platelets. Conditional oligomerization of GP Ib-IX(FKBP)(2) by AP20187, a cell-permeable FKBP dimerizer, caused a decrease in cell translocation velocities on VWF (p < 0.001). Moreover, clustering of GP Ib-IX(FKBP)(2) by AP20187 led to an increase in alpha(IIb)beta(3) function, manifested under static conditions by increased cell adhesion to fibrinogen (p < 0.01) and under flow by increased stable cell adhesion to VWF (p < 0.04). Clustering of GP Ib-IX(FKBP)(2) also stimulated rapid tyrosine phosphorylation of ectopically expressed Syk, a putative downstream effector of GP Ib-IX in platelets. These studies establish that GP Ib-IX oligomerization, per se, affects the interaction of this receptor with VWF and its ability to influence the adhesive function of alpha(IIb)beta(3). By extrapolation, GP Ib-IX clustering in platelets may promote thrombus formation.     

Amino acid residues in the alpha IIb subunit that are critical for ligand binding to integrin alpha IIbbeta 3 are clustered in the beta-propeller model

Several distinct regions of the integrin alpha(IIb) subunit have been implicated in ligand binding. To localize the ligand binding sites in alpha(IIb), we swapped all 27 predicted loops with the corresponding sequences of alpha(4) or alpha(5). 19 of the 27 swapping mutations had no effect on binding to both fibrinogen and ligand-mimetic antibodies (e.g. LJ-CP3), suggesting that these regions do not contain major ligand binding sites. In contrast, swapping the remaining 8 predicted loops completely blocked ligand binding. Ala scanning mutagenesis of these critical predicted loops identified more than 30 discontinuous residues in repeats 2-4 and at the boundary between repeats 4 and 5 as critical for ligand binding. Interestingly, these residues are clustered in the predicted beta-propeller model, consistent with this model. Most of the critical residues are located at the edge of the upper face of the propeller, and several critical residues are located on the side of the propeller domain. None of the predicted loops in repeats 1, 6, and 7, and none of the four putative Ca(2+)-binding predicted loops on the lower surface of the beta-propeller were important for ligand binding. The results map an important ligand binding interface at the edge of the top and on the side of the beta-propeller toroid, centering on repeat 3.     

Agonist-specific structural rearrangements of integrin alpha IIbbeta 3. Confirmation of the bent conformation in platelets at rest and after activation

Concrete structural features of integrin alpha(IIb)beta(3) on the surface of platelets (at rest and after activation) have been obtained from epitope maps based on cross-competition among monoclonal antibodies directed against the alpha(IIb) subunit calf-2 domain and the beta(3) subunit betaA domain of alpha(IIb)beta(3). At rest, the observed intersubunit interface is formed by the sequence stretches beta(3)-(150-216), alpha(IIb) light chain-(1-92), and alpha(IIb) heavy chain-(826-856); and the alpha(IIb) interchain interface is formed by the two latter sequence stretches, disulfide-bonded between alpha(IIb) heavy chain Cys(826) and alpha(IIb) light chain Cys(9). These structural features agree with those observed in the alpha(IIb)beta(3) rudimentary connectivity map in solution and with the alpha(v)beta(3) V-shaped crystal structure (Xiong, J.-P., Zhang, R., Dunker, R., Scott, D. L., Joachimiak, A., Goodman, S. L., and Arnaout, M. A. (2001) Science 294, 339-345), but they disagree with the domain disposition suggested by the actual ultrastructural model. The epitope maps in platelets activated by ADP, thrombin receptor activation peptide, and arachidonic acid differ not only from those in platelets at rest, but also among themselves. The structural rearrangements observed confirm the presence in activated platelets of the crystallographically observed knee and argue against the switchblade mechanism proposed for activation (Beglova, N., Blacklow, S. C., Takagi, J., and Springer, T. A. (2002) Nat. Struct. Biol. 9, 282-287), demonstrate the existence of alpha(IIb)beta(3) agonist-specific activation states, explain the specificity for ligand binding and functional inhibition for some agonists, and predict the existence of agonist-specific final effectors and receptor activation mechanisms. The distinct non-reciprocal competition patterns observed at rest and after activation support the agonist-specific activation states and the existence of intrasubunit and intersubunit allosteric effects, previously proposed as the mechanism for alpha(IIb)beta(3) transmembrane activation.     

Identification of shc as the primary protein binding to the tyrosine-phosphorylated beta 3 subunit of alpha IIbbeta 3 during outside-in integrin platelet signaling

Outside-in signaling mediated by the integrin alpha(IIb)beta(3) (GPIIbIIIa) is critical to platelet function and has been shown to involve the phosphorylation of tyrosine residues on the cytoplasmic tail of beta(3). To identify proteins that bind directly to phosphorylated beta(3), we utilized an affinity column consisting of a peptide modeled on the tyrosine-phosphorylated cytoplasmic domain of beta(3). Tandem mass spectrometric sequencing and immunoblotting demonstrated that Shc was the primary protein binding to phosphorylated beta(3). To determine the involvement of Shc in outside-in alpha(IIb)beta(3) signaling, the phosphorylation of Shc during platelet aggregation was examined; transient Shc phosphorylation was observed when thrombin-stimulated platelets were allowed to aggregate or when aggregation was induced by an LIBS (ligand-induced binding site) antibody, D3. Moreover, Shc was co-immunoprecipitated with tyrosine-phosphorylated beta(3) in detergent lysates of aggregated platelets. Using purified, recombinant protein, it was found that the binding of Shc to monophosphorylated (C-terminal tyrosine) and diphosphorylated beta(3) peptides was direct, demonstrating Shc recognition motifs on phospho-beta(3). Aggregation-induced Shc phosphorylation was also observed to be robust in platelets from wild-type mice, but not in those from mice expressing (Y747F,Y759F) beta(3), which are defective in outside-in alpha(IIb)beta(3) signaling. Thus, Shc is the primary downstream signaling partner of beta(3) in its tyrosine phosphorylation outside-in signaling pathway.     

Galectin-3 regulates integrin alpha2beta1-mediated adhesion to collagen-I and -IV

Galectins are a taxonomically widespread family of galactose-binding proteins of which galectin-3 is known to modulate cell adhesion. Using single cell force spectroscopy, the contribution of galectin-3 to the adhesion of Madin-Darby canine kidney (MDCK) cells to different extracellular matrix proteins was investigated. When adhering to collagen-I or -IV, some cells rapidly entered an enhanced adhesion state, marked by a significant increase in the force required for cell detachment. Galectin-3-depleted cells had an increased probability of entering the enhanced adhesion state. Adhesion enhancement was specific to integrin alpha(2)beta(1), as it was not observed when cells adhered to extracellular matrix substrates by other integrins. The adhesion phenotype of galectin-3-depleted cells was mimicked in a galactoside-deficient MDCK cell line and could be complemented by the addition of recombinant galectin-3. We propose that galectin-3 influences integrin alpha(2)beta(1)-mediated adhesion complex formation by altering receptor clustering.     

Betaig-h3 supports keratinocyte adhesion,migration, and proliferation through alpha3beta1 integrin

betaig-h3 is an extracellular matrix protein and its expression is highly induced by TGF-beta and it has also been suggested to play important roles in skin wound healing. In this paper, we demonstrate that betaig-h3 is present in the papillary layer of dermis and synthesized in the basal keratinocytes in vivo and its expression is induced by TGF-beta in normal human keratinocytes (NHEK) and HaCaT cells. betaig-h3 mediates not only adhesion and spreading of keratinocytes but also supports migration and proliferation. These activities are mediated through interacting with alpha3beta1 integrin. Previously identified two alpha3beta1 integrin-interacting motifs of betaig-h3, EPDIM, and NKDIL, are responsible for these activities. The results suggest that betaig-h3 may regulate keratinocyte functions in normal skin and potentially during wound-healing process.     

Betaig-h3 interacts with alpha3beta1 integrin to promote adhesion and migration of human hepatoma cells

betaig-h3 is a TGF-induced extracellular matrix (ECM) protein. Our previous evidence suggests that beta ig-h3 may promote adhesion and invasion potential of human hepatoma cells, but the mechanism underlying this process is still unknown. The present study identifies a pivotal role for molecules of the beta ig-h3 signal transduction pathway. We demonstrated that beta ig-h3 co-immunoprecipitated with alpha 3beta 1 integrin in human 7721 hepatoma cells. The addition of alpha 3beta 1 integrin antibodies inhibited the elevated adhesion and migration in beta ig-h3-over-expressing 7721 cells, but not in beta ig-h3 siRNA transfected 7721 cells. The expression and activity of integrin downstream molecules FAK and paxillin show a positive correlation with beta ig-h3 expression in different human hepatoma cells. Levels of focal adhesions and stress fibers were decreased in beta ig-h3 siRNA transfected 7721 cells. We suggest that by interaction with alpha 3beta 1 integrin, beta ig-h3 activates FAK-paxillin signaling linkage, leads to cytoskeleton reorganization, and thus enhances the metastatic potentials of human hepatoma cells.     

Distinct ligand binding sites in integrin alpha3beta1 regulate matrix adhesion and cell-cell contact

The integrin alpha3beta1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with alpha3beta1 via a surface loop within the alpha3 beta-propeller (residues 242-246) but outside the laminin binding region, suggesting that uPAR-integrin interactions could signal differently from matrix engagement. To explore this, alpha3-/- epithelial cells were reconstituted with wild-type (wt) alpha3 or alpha3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt alpha3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and gamma-catenin. Src kinase inhibition or expression of Src 1-251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that alpha3beta1 regulates both cell-cell contact and matrix adhesion, but through distinct protein interaction sites within its beta-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition.     

Functions of alpha3beta1 integrin

alpha3beta1 integrin is a laminin receptor with apparently diverse functions. In epithelial cells it acts as a receptor for the basement membrane, whereas in neuronal and possibly tumor cells it mediates migration. Interactions of alpha3beta1 integrin with tetraspanin proteins may provide clues to how it transduces signals that affect cell behavior.     

Ligand-dependent activation of integrin alpha vbeta 3

The ability of leukocytes to self-regulate adhesion during transendothelial and extravascular migration is fundamental to the performance of immune surveillance in complex extracellular matrices. Leukocyte adhesion is regulated through the modulation of integrin receptors such as alpha(v)beta(3). In this study, we examined the activation of alpha(v)beta(3) resulting from attachment to vitronectin or fibronectin. In K562 cells stably expressing transfected alpha(v)beta(3), adhesion to vitronectin required tyrosine phosphorylation of the beta(3) subunit and activation of phosphoinositide 3-kinase and protein kinase C. In contrast, adhesion to fibronectin proceeded without beta(3)-tyrosine phosphorylation or the activities of phosphoinositide 3-kinase or protein kinase C. Firm adhesion to both ligands and actin stress fiber formation required both Syk and Rho activity, suggesting that each ligand employs unique signaling pathways to achieve an active integrin complex, likely merging at a common RhoGEF such as Vav. Distinct signaling by a single integrin species interacting with different ligands permits initiation of additional cellular processes specific to the current task and provides an explanation for what has been described as promiscuous ligand specificity among integrins.     

The cAMP-Epac-Rap1 pathway regulates cell spreading and cell adhesion to laminin-5 through the alpha3beta1 integrin but not the alpha6beta4 integrin

Laminin-5 is an important constituent of the basal lamina. The receptors for laminin-5, the integrins alpha3beta1 and alpha6beta4, have been associated with epithelial wound migration and carcinoma invasion. The signal transduction mechanisms that regulate these integrins are not well understood. We report here that the small GTPase Rap1 regulates the adhesion of a number of cell lines to various extracellular matrix proteins including laminin-5. cAMP also mediates cell adhesion and spreading on laminin-5, a process that is independent of protein kinase A but rather dependent on Epac1, a cAMP-dependent exchange factor for Rap. Interestingly, although both alpha3beta1 and alpha6beta4 mediate adhesion to laminin-5, only alpha3beta1-dependent adhesion is dependent on Rap1. These results provide evidence for a function of the cAMP-Epac-Rap1 pathway in cell adhesion and spreading on different extracellular matrix proteins. They also define different roles for the laminin-binding integrins in regulated cell adhesion and subsequent cell spreading.