rho family GTPase activating proteins p190, bcr and rhoGAP show distinct specificities in vitro and in vivo.

rho family GTPases link extracellular signals to changes in the organization of cytoskeletal actin. Serum stimulation of quiescent Swiss 3T3 fibroblasts leads to rho-dependent actin stress fibre formation and focal adhesions, whilst several growth factors initiate signalling pathways leading to rac-dependent actin polymerization at the plasma membrane, and membrane ruffling. The product of the breakpoint cluster region gene bcr, rho GTPase accelerating protein (rhoGAP) and rasGAP-associated p190 share structurally related rho GAP domains, and possess GAP activity for rho family members in vitro. We have directly compared the activities of the isolated GAP domains of these three proteins in regulating different rho family GTPases, both by in vitro assays and by microinjection, to address their possible physiologic functions. We show that bcr accelerates the GTPase activity of rac, but not rho in vitro, and inhibits rac-mediated membrane ruffling, but not rho-mediated stress fibre formation, after microinjection into Swiss 3T3 fibroblasts. In vitro, rhoGAP has a striking preference for G25K as a substrate, whilst p190GAP has marked preferential activity for rho. Furthermore, p190 preferentially inhibits rho-mediated stress fibre formation in vivo. Our data suggest that p190, rhoGAP and bcr play distinct roles in signalling pathways mediated through different rho family GTPases.     

PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading

BACKGROUND: It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. RESULTS: The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. CONCLUSIONS: We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.     

Bidirectional signaling between the cytoskeleton and integrins

Clustering of integrins into focal adhesions and focal complexes is regulated by the actin cytoskeleton. In turn, actin dynamics are governed by Rho family GTPases. Integrin-mediated adhesion activates these GTPases, triggering assembly of filopodia, lamellipodia and stress fibers. In the past few years, signaling pathways have begun to be identified that promote focal adhesion disassembly and integrin dispersal. Many of these pathways result in decreased myosin-mediated cell contractility.     

Integrins and cell signaling in chondrocytes

Integrins are adhesion receptor heterodimers that transmit information from the extracellular matrix (ECM) to the cell through activation of cell signaling pathways. Chondrocytes express several members of the integrin family including alpha5beta1 which is the primary chondrocyte receptor for fibronectin. Cell signaling mediated through integrins regulates several chondrocyte functions including differentiation, matrix remodeling, responses to mechanical stimulation and cell survival. Integrin-mediated activation of members of the mitogen-activated protein kinase family likely plays a key role in transmitting signals regulating chondrocyte gene expression. Upstream mediators of mitogen-activated protein kinase (MAP kinase) activation include focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (pyk2) which are both expressed by chondrocytes. A better understanding of chondrocyte integrin signaling is needed to define the mechanisms by which the ECM regulates chondrocyte function.     

Novel CD47-dependent intercellular adhesion modulates cell migration

CD47 is a ubiquitously expressed plasma membrane protein, also known as Integrin Associated Protein, that modulates cell adhesion both through alteration of the avidity of integrin binding and through interaction with its own ligands, the extracellular matrix protein thrombospondin (TSP) and the plasma membrane response regulator SIRPalpha1. We now show that CD47 expression on fibroblasts can induce intercellular adhesion resulting in cell aggregation in the absence of active integrins, SIRPalpha1 binding, and detectable TSP. CD47-expressing cells preferentially bind to other CD47-expressing cells, and intercellular adhesion requires stimulation by serum or a CD47-binding peptide from TSP. Cell-cell adhesion is inhibited by pertussis toxin and C. difficile toxin B, and both adherent and aggregating CD47-expressing fibroblasts have more rac in the GTP bound state than CD47-deficient cells. Spontaneous migration of Jurkat lymphocytes through a fibroblast monolayer is decreased by fibroblast expression of CD47, consistent with an increased barrier function of the CD47 expressing cells. The lymphocyte chemoattractant SDF-1alpha stimulates migration of Jurkat cells through this monolayer only if both the lymphocytes and fibroblasts express CD47, and the inhibition of migration by a CD47-interacting peptide from TSP similarly requires CD47 expression on both cell types. Thus, signaling dependent on both heterotrimeric and rho family GTPases can induce CD47 to participate in cell-cell interactions independent of known ligands that enhance intercellular adhesion and modulate cell migration.     

A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling

The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with beta1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with beta1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes beta1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, beta1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with beta1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal beta1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.     

Members of the Zyxin family of LIM proteins interact with members of the p130Cas family of signal transducers.

Integrin binding to extracellular matrix proteins induces formation of signaling complexes at focal adhesions. Zyxin co-localizes with integrins at sites of cell-substratum adhesion and is postulated to serve as a docking site for the assembly of multimeric protein complexes involved in regulating cell motility. Recently, we identified a new member of the zyxin family called TRIP6. TRIP6 is localized at focal adhesions and overexpression of TRIP6 slows cell migration. In an effort to define the molecular mechanism by which TRIP6 affects cell migration, the yeast two-hybrid assay was employed to identify proteins that directly bind to TRIP6. This assay revealed that both TRIP6 and zyxin interact with CasL/HEF1, a member of the Cas family. This association is mediated by the LIM region of the zyxin family members and the SH2 domain-binding region of CasL/HEF1. Furthermore, the association between p130(Cas) and the two zyxin family members was demonstrated to occur in vivo by co-immunoprecipitation. Zyxin and Cas family members may cooperate to regulate cell motility.     

ERK1 associates with alpha(v)beta 3 integrin and regulates cell spreading on vitronectin

Kinases that associate with integrins are likely to mediate the assembly/disassembly of cell:matrix junctions during cell migration. Here we show that ERK1 associates with alpha(v)beta(3) integrin following the addition of platelet-derived growth factor to serum-starved Swiss or NIH 3T3 fibroblasts in an interaction that is mediated by the central region of the beta(3) integrin cytodomain. alpha(v)beta(3).ERK1 association occurred prior to focal complex formation and was seen to initiate in small punctate complexes primarily in the peripheral regions of the plasma membrane. Expression of a dominant negative mutant of ERK1 (but not ERK2) significantly reduced the spreading of cells on vitronectin, whereas cell spreading on fibronectin was unaffected by inhibition of ERK1. In contrast, inhibition of ERK activation by PD98059 had no effect on the platelet-derived growth factor-regulated Rab4-dependent flux of alpha(v)beta(3) integrin from early endosomes to the plasma membrane, an event that is also necessary for cells to spread efficiently on vitronectin. We propose that alpha(v)beta(3) integrin must recycle to the plasma membrane via the Rab4 pathway and recruit active ERK1 in order to function efficiently.     

Requirement for Rho in Integrin Signalling

Overnight culture of Swiss 3T3 cells in serum-free medium leads to loss of focal adhesions and associated actin stress fibres, although the cells remain well spread. The small GTP-binding protein Rho is required for the formation of stress fibres and focal adhesions induced by growth factors such as lysophosphatidic acid (LPA) in serum-starved Swiss 3T3 cells, and for the LPA-induced tyrosine phosphorylation of several focal adhesion proteins. Plating of cells on extracellular matrix proteins also stimulates protein tyrosine phosphorylation and the formation of stress fibres and focal adhesions in the absence of added growth factors. These responses were inhibited in cells scrape-loaded with the Rho inhibitor C3 transferase. Focal adhesion and stress fibre formation was also triggered by addition of a peptide GRGDS, which is recognised by a number of integrins and is contained within the cell binding domain of a variety of extracellular matrix proteins. The activity of the GRGDS peptide was blocked by microinjecting cells with C3 transferase, suggesting that peptide binding to integrins stimulates a Rho-dependent assembly of focal adhesions. These experiments indicate that Rho is involved in signalling downstream of integrins.     

Integrin dynamics and matrix assembly: tensin-dependent translocation of alpha(5)beta(1) integrins promotes early fibronectin fibrillogenesis

Fibronectin matrix assembly is a multistep, integrin-dependent process. To investigate the role of integrin dynamics in fibronectin fibrillogenesis, we developed an antibody-chasing technique for simultaneous tracking of two integrin populations by different antibodies. We established that whereas the vitronectin receptor alpha(v)beta(3) remains within focal contacts, the fibronectin receptor alpha(5)beta(1) translocates from focal contacts into and along extracellular matrix (ECM) contacts. This escalator-like translocation occurs relative to the focal contacts at 6.5 +/- 0.7 microm/h and is independent of cell migration. It is induced by ligation of alpha(5)beta(1) integrins and depends on interactions with a functional actin cytoskeleton and vitronectin receptor ligation. During cell spreading, translocation of ligand-occupied alpha(5)beta(1) integrins away from focal contacts and along bundles of actin filaments generates ECM contacts. Tensin is a primary cytoskeletal component of these ECM contacts, and a novel dominant-negative inhibitor of tensin blocked ECM contact formation, integrin translocation, and fibronectin fibrillogenesis without affecting focal contacts. We propose that translocating alpha(5)beta(1) integrins induce initial fibronectin fibrillogenesis by transmitting cytoskeleton-generated tension to extracellular fibronectin molecules. Blocking this integrin translocation by a variety of treatments prevents the formation of ECM contacts and fibronectin fibrillogenesis. These studies identify a localized, directional, integrin translocation mechanism for matrix assembly.     

Platelet-derived growth factor inhibits smooth muscle cell adhesion to fibronectin by ERK-dependent and ERK-independent pathways

The adhesion of cells to the extracellular matrix plays a major role in cell migration. Pretreatment with platelet-derived growth factor (PDGF) inhibited the adhesion of smooth muscle cells to fibronectin by 80%. This inhibition decreased as concentrations of fibronectin increased. In the presence of 200 microm GRGDS peptide, only 45% of PDGF-treated cells adhered to fibronectin compared with 80% of control cells. This indicates that a decrease in integrin avidity was induced by PDGF. Cell adhesion was partially restored when the activation of the extracellular signal-regulated kinase (ERK) was inhibited with PD98059. The remaining inhibition of adhesion (50%) was independent of the fibronectin concentration, suggesting that the ERK pathway is involved in the decrease in integrin avidity. This was confirmed by depleting ERK protein levels by treatment with ERK antisense oligonucleotide. The adhesion of ERK control oligonucleotide-treated cells decreased by 41% when the concentration of GRGDS peptide was increased from 50 to 200 microm but only decreased by 11% in ERK antisense oligonucleotide-treated cells. Treatment with PDGF also delayed focal complex assembly and inhibited stress fiber formation. Consistent with a delay in tyrosine phosphorylation of paxillin, PDGF treatment caused a lag in focal complex formation, although this was not associated with any change in Src family tyrosine kinase activity. Our results indicate that PDGF inhibits smooth muscle cells adhesion by two pathways. The first involves an ERK-dependent decrease in integrin avidity; the second involves the ERK-independent inhibition of focal complex assembly.     

Integrin-mediated signal transduction pathways.

Integrins serve as adhesion receptors for extracellular matrix proteins and also transduce biochemical signals into the cell. They regulate a variety of cellular functions, including spreading, migration, proliferation and apoptosis. Many signaling pathways downstream of integrins have been identified and characterized and are discussed here. In particular, integrins regulate many protein tyrosine kinases and phosphatases, such as FAK and Src, to coordinate many of the cell processes mentioned above. The regulation of MAP kinases by integrins is important for cell growth or other functions, and the putative roles of Ras and FAK in these pathways are discussed. Phosphatidylinositol lipids and their modifying enzymes, particularly PI 3-kinase, are strongly implicated as mediators of integrin-regulated cytoskeletal changes and cell migration. Similarly, actin cytoskeleton regulation by the Rho family of GTPases is coordinated with integrin signaling to regulate cell spreading and migration, although the exact relationship between these pathways is not clear. Finally, intracellular pH and calcium fluxes by integrins are suggested to affect a variety of cellular proteins and functions.     

Integrins uncouple Src-induced morphological and oncogenic transformation

Expression of activated mutants of c-Src in epithelial cells can induce tumorigenicity. In addition to such oncogenic transformation, the cells undergo a dramatic morphological transformation: cell-cell contacts are disrupted, spreading on extracellular matrix proteins is suppressed, actin stress fibers and focal contacts are lost, and podosomes are formed. We have previously shown that integrin alphavbeta3 strongly supports Src-mediated oncogenic transformation through an interaction at the beta3 cytoplasmic tail. Our current findings demonstrate that this interaction does not affect Src-mediated morphological alterations, thus separating oncogenic from morphological transformation. Moreover, beta1 and beta3 integrins differently affect the various aspects of Src-induced morphological transformation. High levels of beta3, but not beta1, integrins can prevent Src-induced cell rounding although stress fiber disassembly and podosome formation still occur. Studies using chimeric integrin subunits demonstrate that this protection requires the beta3 extracellular domain. Finally, like tumor formation, podosome assembly occurs independent of beta3 phosphorylation. Instead, phosphorylation of beta1 is required to suppress Rho-mediated contractility in order to assemble podosomes. Thus, integrins regulate Src-mediated oncogenic transformation and various aspects of morphological transformation through dissociable pathways.     

Fibronectin, integrins, and growth control

Cell proliferation is controlled not only by soluble mitogens but also by components of the extracellular matrix (ECM) such as fibronectin, to which cells adhere via the integrin family of transmembrane receptors. Input from both growth factor receptors and integrins is required to stimulate progression through the G1 phase of the cell cycle, via induction of G1 cyclins and suppression of inhibitors of the G1 cyclin-dependent kinases. Extensive crosstalk takes place between integrin and growth factor receptor signaling pathways, and mitogenic signaling is weak and transient in the absence of integrin-mediated cell adhesion. In normal untransformed cells, all of the important mitogenic signal transduction cascades, namely those downstream of the Ras and Rho family small GTPases and the phosphoinositide 3-OH kinase-PKB/Akt pathway, are regulated by integrin-mediated cell adhesion. As a result, these cells are anchorage-dependent for growth. In contrast, constitutive activity of each of these pathways has been reported in cancer cells, which not only reduces their mitogen dependence but also allows these cells to grow in an anchorage-independent fashion.     

Coordinate signaling by integrins and receptor tyrosine kinases in the regulation of G1 phase cell-cycle progression

Cell proliferation is dependent upon the activation of receptor tyrosine kinases and integrins by soluble growth factors and extracellular matrix proteins, respectively. It is now apparent that concerted, rather than individual, signaling by these receptors is the critical feature responsible for cell-cycle progression through G1 phase. ERK (extracellular signal-regulated kinase), Rho GTPases and G1-phase cyclin-dependent kinases are all regulated jointly by growth-factor receptors and integrins. Recent studies have begun to reveal how this regulated signaling in the cytoplasm is linked to activation of the G1-phase cyclin-dependent kinases in the nucleus.     

RSK2 Protein Suppresses Integrin Activation and Fibronectin Matrix Assembly and Promotes Cell Migration

Modulation of integrin activation is important in many cellular functions including adhesion, migration, and assembly of the extracellular matrix. RSK2 functions downstream of Ras/Raf and promotes tumor cell motility and metastasis. We therefore investigated whether RSK2 affects integrin function. We report that RSK2 mediates Ras/Raf inactivation of integrins. As a result, we find that RSK2 impairs cell adhesion and integrin-mediated matrix assembly and promotes cell motility. Active RSK2 appears to affect integrins by reducing actin stress fibers and disrupting focal adhesions. Moreover, RSK2 co-localizes with the integrin activator talin and is present at integrin cytoplasmic tails. It is thereby in a position to modulate integrin activation and integrin-mediated migration. Activation of RSK2 promotes filamin phosphorylation and binding to integrins. We also find that RSK2 is activated in response to integrin ligation to fibronectin. Thus, RSK2 could participate in a feedback loop controlling integrin function. These results reveal RSK2 as a key regulator of integrin activity and provide a novel mechanism by which it may promote cell migration and cancer metastasis.     

Focal adhesion kinase facilitates platelet-derived growth factor-BB-stimulated ERK2 activation required for chemotaxis migration of vascular smooth muscle cells

The focal adhesion (FAK) non-receptor protein-tyrosine kinase (PTK) links both extracellular matrix/integrin and growth factor stimulation to intracellular signals promoting cell migration. Here we show that both transient and stable overexpression of the FAK C-terminal domain termed FRNK (FAK-related non-kinase) inhibits serum and platelet-derived growth factor (PDGF)-BB-induced vascular smooth muscle cell (SMC) migration in wound healing and in vitro Boyden Chamber chemotaxis assays, respectively. Expression of FRNK, but not a point mutant of FRNK (FRNK L1034S), disrupted the formation of a complex containing both FAK and the activated PDGF-beta receptor and resulted in reduced tyrosine phosphorylation of endogenous FAK at the Tyr-397 binding site for Src family PTKs. As demonstrated using FAK-deficient and FAK-reconstituted fibroblasts, FAK positively contributed to PDGF-BB-stimulated ERK2/MAP kinase activity, and in SMCs, ERK2/MAP kinase activity was required for PDGF-BB-stimulated chemotaxis. Stable expression of FRNK but not FRNK L1034S expression in SMCs lowered the extent and duration of stimulated ERK2/MAP kinase activation at low but not at high PDGF-BB concentrations. Importantly, stable expression of FRNK in SMCs did not affect SMC morphology or proliferation in culture. Because the increased migration of vascular SMCs in response to extracellular matrix proteins and growth factors contributes to neointima formation, our results show that FAK inhibition by FRNK expression may provide a novel approach to regulate abnormal vascular SMC migration in vivo.     

Mapping of heparin/heparan sulfate binding sites on αvβ3 integrin by molecular docking

Heparin/heparan sulfate interact with growth factors, chemokines, extracellular proteins, and receptors. Integrins are αβ heterodimers that serve as receptors for extracellular proteins, regulate cell behavior, and participate in extracellular matrix assembly. Heparin binds to RGD‐dependent integrins (αIIbβ3, α5β1, αvβ3, and αvβ5) and to RGD‐independent integrins (α4β1, αXβ2, and αMβ2), but their binding sites have not been located on integrins. We report the mapping of heparin binding sites on the ectodomain of αvβ3 integrin by molecular modeling. The surface of the ectodomain was scanned with small rigid probes mimicking the sulfated domains of heparan sulfate. Docking results were clustered into binding spots. The best results were selected for further docking simulations with heparin hexasaccharide. Six potential binding spots containing lysine and/or arginine residues were identified on the ectodomain of αvβ3 integrin. Heparin would mostly bind to the top of the genu domain, the Calf‐I domain of the α subunit, and the top of the β subunit of RGD‐dependent integrins. Three spots were close enough from each other on the integrin surface to form an extended binding site that could interact with heparin/heparan sulfate chains. Because heparin does not bind to the same integrin site as protein ligands, no steric hindrance prevents the formation of ternary complexes comprising the integrin, its protein ligand, and heparin/heparan sulfate. The basic amino acid residues predicted to interact with heparin are conserved in the sequences of RGD‐dependent but not of RGD‐independent integrins suggesting that heparin/heparan sulfate could bind to different sites on these two integrin subfamilies. Copyright © 2013 John Wiley & Sons, Ltd.     

Integrin activation and focal complex formation in cardiac hypertrophy

Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.