Close homolog of L1 is an enhancer of integrin-mediated cell migration

Close homolog of L1 (CHL1) is a member of the L1 family of cell adhesion molecules expressed by subpopulations of neurons and glia in the central and peripheral nervous system. It promotes neurite outgrowth and neuronal survival in vitro. This study describes a novel function for CHL1 in potentiating integrin-dependent cell migration toward extracellular matrix proteins. Expression of CHL1 in HEK293 cells stimulated their haptotactic migration toward collagen I, fibronectin, laminin, and vitronectin substrates in Transwell assays. CHL1-potentiated cell migration to collagen I was dependent on alpha1beta1 and alpha2beta1 integrins, as shown with function blocking antibodies. Potentiated migration relied on the early integrin signaling intermediates c-Src, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Enhancement of migration was disrupted by mutation of a potential integrin interaction motif Asp-Gly-Glu-Ala (DGEA) in the sixth immunoglobulin domain of CHL1, suggesting that CHL1 functionally interacts with beta1 integrins through this domain. CHL1 was shown to associate with beta1 integrins on the cell surface by antibody-induced co-capping. Through a cytoplasmic domain sequence containing a conserved tyrosine residue (Phe-Ile-Gly-Ala-Tyr), CHL1 recruited the actin cytoskeletal adapter protein ankyrin to the plasma membrane, and this sequence was necessary for promoting integrin-dependent migration to extracellular matrix proteins. These results support a role for CHL1 in integrin-dependent cell migration that may be physiologically important in regulating cell migration in nerve regeneration and cortical development.     

Cadherin-related neuronal receptor 1 (CNR1) has cell adhesion activity with beta1 integrin mediated through the RGD site of CNR1

Cadherin-related neuronal receptor (CNR) proteins are a diverse set of synaptic protocadherins, but little is known about its adhesive properties. We found that overexpressed CNR1 protein localized on the cell surface of HEK293T cells and increased the calcium-dependent cell aggregation potential. However, we could not detect the strong homophilic binding activity of CNR1 EC-Fc fusion protein in vitro. Parental HEK293T cells adhered to Arg-Gly-Asp (RGD) motif of EC1 domain of CNR1-Fc fusion protein. The fusion protein that the Asp73 of EC1 point-mutated to Glu (RGE-Fc) lost the adhesive activity. The adhesion activity of HEK293T cells to CNR1 EC-Fc fusion protein was completely blocked by inhibitors of integrins, including RGDS peptide and anti-beta1 integrin antibodies. The increased cell-aggregative property of CNR1 transfectants was also blocked by RGDS peptides. At cell-cell junctions of the CNR1 transfectants, co-localization between CNR1 and HEK293T endogenous beta1 integrin was observed. Furthermore, the spatiotemporal expression patterns of CNR and beta1 integrin nearly overlapped in the molecular layer of the developing mouse cerebellum in the main stage of synaptogenesis. These results indicate that CNR1 has a heterophilic, calcium-dependent cell adhesion activity with the beta1 integrin subfamily, and raise the possibility of CNR-beta1 integrin association in synaptogenesis.     

Integrin alpha(v)beta1 is an adenovirus coreceptor

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor alpha(v)beta3 or alpha(v)beta5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin alpha(v)beta1. Function-blocking antibodies directed against alpha(v) or beta1, but not beta3, beta5, or alpha5, integrin subunits block Ad infection and viral endocytosis. Therefore, alpha(v)beta1 serves as a coreceptor for Ad infection, and the lack of beta3 and/or beta5 but the relatively high expression of alpha(v)beta1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.     

A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5

The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.     

Urokinase receptors promote beta1 integrin function through interactions with integrin alpha3beta1

The urokinase receptor (uPAR) is linked to cellular migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate cell signaling in response to urokinase (uPA) binding. The mechanisms for these activities remain incompletely defined, although uPAR was recently identified as a cis-acting ligand for the beta2 integrin CD11b/CD18 (Mac-1). Here we show that a major beta1 integrin partner for uPAR/uPA signaling is alpha3. In uPAR-transfected 293 cells uPAR complexed (>90%) with alpha3beta1 and antibodies to alpha3 blocked uPAR-dependent vitronectin (Vn) adhesion. Soluble uPAR bound to recombinant alpha3beta1 in a uPA-dependent manner (K(d) < 20 nM) and binding was blocked by a 17-mer alpha3beta1 integrin peptide (alpha325) homologous to the CD11b uPAR-binding site. uPAR colocalized with alpha3beta1 in MDA-MB-231 cells and uPA (1 nM) enhanced spreading and focal adhesion kinase phosphorylation on fibronectin (Fn) or collagen type I (Col) in a pertussis toxin- and alpha325-sensitive manner. A critical role of alpha3beta1 in uPA signaling was verified by studies of epithelial cells from alpha3-deficient mice. Thus, uPAR preferentially complexes with alpha3beta1, promoting direct (Vn) and indirect (Fn, Col) pathways of cell adhesion, the latter a heterotrimeric G protein-dependent mechanism of signaling between alpha3beta1 and other beta1 integrins.     

The integrin beta1 subunit cytoplasmic tail forms oligomers: a potential role in beta1 integrin clustering

Integrins are alpha/beta heterodimeric cell surface receptors devoid of enzymatic activity. Signal transduction therefore requires the association of cytosolic and cytoskeletal proteins with the integrin subunit intracellular regions. This association is initiated upon ligand binding to the integrin receptor and includes clustering of the integrins and recruitment of focal adhesion-associated proteins. Whether integrin clustering is solely dependent on ligand binding to the integrin extracellular parts or involves also interactions between the intracellular tails of integrins is so far unknown. To investigate intracellular events in integrin clustering, we have used peptides corresponding to the integrin beta1 cytoplasmic region. Loading of cells with the peptides results in a decreased cell adhesion and in an inhibition of cell spreading in agreement with the previously reported dominant negative effect of the beta1 integrin cytoplasmic tail on integrin clustering. Direct protein-protein interaction studies by surface plasmon resonance demonstrate that integrin beta1 cytoplasmic peptides self-associate in contrast to integrin beta3 cytoplasmic tails. Size exclusion chromatography and SDS-PAGE analysis of the peptides further show that the integrin beta1 cytoplasmic parts form oligomers and that they assume alpha helical conformation to the extent of about 13% and that this fraction is increased upon aggregation. Thus self-association of the integrin beta1 subunit cytoplasmic regions may be central to beta1 integrin clustering.     

Alpha 4 beta 1 integrin/VCAM-1 interaction activates alpha L beta 2 integrin-mediated adhesion to ICAM-1 in human T cells

Modulation of integrin affinity and/or avidity provides a regulatory mechanism by which leukocyte adhesion to endothelium is strengthened or weakened at different stages of emigration. In this study, we demonstrate that binding of high-affinity alpha 4 beta 1 integrins to VCAM-1 strengthens alpha L beta 2 integrin-mediated adhesion. The strength of adhesion of Jurkat cells, a human leukemia T cell line, or MnCl2-treated peripheral blood T cells to immobilized chimeric human VCAM-1/Fc, ICAM-1/Fc, or both was quantified using parallel plate flow chamber leukocyte detachment assays in which shear stress was increased incrementally (0.5-30 dynes/cm2). The strength of adhesion to VCAM-1 plus ICAM-1, or to a 40-kDa fragment of fibronectin containing the CS-1 exon plus ICAM-1, was greater than the sum of adhesion to each molecule alone. Treatment of Jurkat or blood T cells with soluble cross-linked VCAM-1/Fc or HP2/1, a mAb to alpha 4, significantly increased adhesion to ICAM-1. These treatments induced clustering of alpha L beta 2 integrins, but not the high-affinity beta 2 integrin epitope recognized by mAb 24. Up-regulated adhesion to ICAM-1 was abolished by cytochalasin D, an inhibitor of cytoskeletal rearrangement. Taken together, our data suggest that the binding of VCAM-1 or fibronectin to alpha 4 beta 1 integrins initiates a signaling pathway that increases beta 2 integrin avidity but not affinity. A role for the cytoskeleton is implicated in this process.     

EWI-2 regulates alpha3beta1 integrin-dependent cell functions on laminin-5

EWI-2, a cell surface immunoglobulin SF protein of unknown function, associates with tetraspanins CD9 and CD81 with high stoichiometry. Overexpression of EWI-2 in A431 epidermoid carcinoma cells did not alter cell adhesion or spreading on laminin-5, and had no effect on reaggregation of cells plated on collagen I (alpha2beta1 integrin ligand). However, on laminin-5 (alpha3beta1 integrin ligand), A431 cell reaggregation and motility functions were markedly impaired. Immunodepletion and reexpression experiments revealed that tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin, but not to other integrins. CD81 also controlled EWI-2 maturation and cell surface localization. EWI-2 overexpression not only suppressed cell migration, but also redirected CD81 to cell filopodia and enhanced alpha3beta1-CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance alpha3beta1-CD81 complex formation. These results show how laterally associated EWI-2 might regulate alpha3beta1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes.     

TGF-beta 1 modulated the expression of alpha 5 beta 1 integrin and integrin-mediated signaling in human hepatocarcinoma cells

Integrins are a family of cell surface adhesion molecules which mediate cell adhesion and initiate signaling pathways that regulate cell spreading, migration, differentiation, and proliferation. TGF-beta is a multifunctional factor that induces a wide variety of cellular processes. In this study, we show that, TGF-beta 1 treatment enhanced the amount of alpha 5 beta 1 integrin on cell surface, the mRNA level of alpha 5 subunit, and subsequently stimulated cell adhesion onto a fibronectin (Fn) and laminin (Ln) matrix in SMMC-7721 cells. TGF-beta 1 could also promote cell migration. Furthermore, our results showed that TGF-beta1 treatment stimulated the tyrosine phosphorylation level of FAK, which can be activated by the ligation and clustering of integrins. PTEN can directly dephosphorylate FAK, and the results that TGF-beta 1 could down-regulate PTEN at protein level suggested that TGF-beta 1 might stimulate FAK phosphorylation through increasing integrin signaling and reducing dephosphorylation of FAK. These studies indicated that TGF-beta 1 and integrin-mediated signaling act synergistically to enhance cell adhesion and migration and affect downstream signaling molecules of hepatocarcinoma cells.     

Transforming growth factors beta(1) (TGF-beta(1)) and TGF-beta(2) promote glioma cell migration via Up-regulation of alpha(V)beta(3) integrin expression

The migratory behaviour of malignant gliomas relies on the interaction of integrins with extracellular matrix (ECM) components. Transforming growth factor-beta(1) (TGF-beta(1)) potently stimulates glioma cell motility whereas TGF-beta(2) is known for its immunosuppressive properties. Here, we show that both TGF-beta(1) and TGF-beta(2) promote migration of glioma cells. In parallel, TGF-beta(1) and TGF-beta(2) induce alpha(V) and beta(3) intergrin mRNA expression and enhance cell surface expression of alpha(V)beta(3) integrin. TGF-beta-mediated promotion of migration is abrogated by echistatin, a Arg-Gly-Asp (RGD) peptide antagonist of alpha(V)beta(3) integrin, and by a neutralizing anti-alpha(V)beta(3) integrin antibody. Taken together, we report a novel mechanism by which TGF-beta modulates cell ECM interactions and promotes glioma cell motility.     

Different integrins mediate cell spreading, haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin

Collaborative role of various fibronectin-binding integrins (alpha5beta1, alphavbeta1 and alphavbeta6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of alphavbeta6 integrin was strongly and specifically upregulated by transforming growth factor-beta1 (TGFbeta1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFbeta1. Based on antibody blocking experiments, both untreated and TGFbeta1-treated HaCaT cells used alphavbeta6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFbeta1-treated cells, the untreated cells also needed alpha5beta1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFbeta1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on alphavbeta6 integrin, while alphavbeta1 and alpha5beta1 integrins played a lesser role both in untreated and TGFbeta1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by beta1 integrins, and alphavbeta6 integrin showed a minor role. The migration process appeared to involve a number of beta1 integrins that could adaptively replace each other when blocking antibodies were present. Thus, keratinocytes appear to use different fibronectin receptors for different functions, such as cell spreading, haptotaxis and lateral migration. The cells can also adapt to a situation where one receptor is unfunctional by switching to another receptor of the same ligand.     

The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility

Integrin affinity is modulated by intracellular signaling cascades, in a process known as "inside-out" signaling, leading to changes in cell adhesion and motility. Protein kinase C (PKC) plays a critical role in integrin-mediated events; however, the mechanism that links PKC to integrins remains unclear. Here, we report that PKCepsilon positively regulates integrin-dependent adhesion, spreading, and motility of human glioma cells. PKCepsilon activation was associated with increased focal adhesion and lamellipodia formation as well as clustering of select integrins, and it is required for phorbol 12-myristate 13-acetate-induced adhesion and motility. We provide novel evidence that the scaffolding protein RACK1 mediates the interaction between integrin beta chain and activated PKCepsilon. Both depletion of RACK1 by antisense strategy and overexpression of a truncated form of RACK1 which lacks the integrin binding region resulted in decreased PKCepsilon-induced adhesion and migration, suggesting that RACK1 links PKCepsilon to integrin beta chains. Altogether, these results provide a novel mechanistic link between PKC activation and integrin-mediated adhesion and motility.     

Beta 1 and beta 3 integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix.

Extracellular matrix receptors on ductus arteriosus smooth muscle cells (SMC) must enable the cells to migrate through both interstitial and basement membrane matrices to form intimal mounds during postnatal ductus closure. We examined the role of beta 1 and beta 3 integrin receptors on SMC adhesion and migration. Using a new assay to measure cell migration, we found that lamb ductus arteriosus SMC attach to and migrate over surfaces coated with fibronectin (FN), laminin (LN), vitronectin (VN), and collagens I (I) and IV (IV). Blocking antibodies, specific to different integrin complexes, showed that SMC adhesion to FN, LN, I, and IV depended exclusively on functioning beta 1 integrins with little, if any, contribution by the alpha V beta 3 integrin; on the other hand, cell migration over these substrates depended to a large extent on the alpha V beta 3 receptor. Immunofluorescent staining demonstrated that during the early phase of SMC migration, the beta 1 integrins organized rapidly into focal plaques that, with time, gradually covered the cell's basal surface; on the other hand, the beta 3 receptor remained concentrated at all times at the cell's margins. Ligand affinity chromatography and immunoprecipitation techniques identified a unique series of beta 1 integrins binding to each matrix component: FN (alpha 5 beta 1, alpha 3 beta 1, alpha V beta 1), LN (alpha 1 beta 1, alpha 7 beta 1), VN (alpha V beta 1), I (alpha 1 beta 1, alpha 2 beta 1), and IV (alpha 1 beta 1). In contrast, the beta 3 integrin, alpha V beta 3, bound to all the substrates tested: FN, LN, VN, I, and IV. The results indicate that beta 1 and beta 3 integrins may play different roles in attachment and migration as SMC move through the vascular extracellular matrix to produce obliteration of the ductus arteriosus lumen.     

Alpha1B-adrenoceptor signaling and cell motility: GTPase function of Gh/transglutaminase 2 inhibits cell migration through interaction with cytoplasmic tail of integrin alpha subunits

A multifunctional enzyme, G(h), is a GTP-binding protein that couples to the alpha(1B)-adrenoreceptor and stimulates phospholipase C-delta1 but also displays transglutaminase 2 (TG2) activity. G(h)/TG2 has been implicated to play a role in cell motility. In this study we have examined which function of G(h)/TG2 is involved in this cellular response and the molecular basis. Treatment of human aortic smooth muscle cell with epinephrine inhibits migration to fibronectin and vitronectin, and the inhibition is blocked by the alpha(1)-adrenoreceptor antagonist prazosin or chloroethylclonidine. Up-regulation or overexpression of G(h)/TG2 in human aortic smooth muscle cells, DDT1-MF2, or human embryonic kidney cells, HEK 293 cells, results in inhibition of the migratory activity, and stimulation of the alpha(1B)-adrenoreceptor with the alpha(1) agonist further augments the inhibition of migration of human aortic smooth muscle cells and DDT1-MF2. G(h)/TG2 is coimmunoprecipitated by an integrin alpha(5) antibody and binds to the cytoplasmic tail peptide of integrins alpha(5), alpha(v), and alpha(IIb) subunits in the presence of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). Mutation of Lys-Arg residues in the GFFKR motif, present in the alpha(5)-tail, significantly reduces the binding of GTPgammaS-G(h)/TG2. Moreover, the motif-containing integrin alpha(5)-tail peptides block G(h)/TG2 coimmunoprecipitation and reverse the inhibition of the migratory activity of HEK 293 cells caused by overexpression G(h)/TG2. These results provide evidence that G(h) function initiates the modulation of cell motility via association of GTP-bound G(h)/TG2 with the GFFKR motif located in integrin alpha subunits.     

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.     

Paxillin binding to the alpha 4 integrin subunit stimulates LFA-1 (integrin alpha L beta 2)-dependent T cell migration by augmenting the activation of focal adhesion kinase/proline-rich tyrosine kinase-2

Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1).     

Disruption of focal adhesions by integrin cytoplasmic domain-associated protein-1 alpha

Regulation of integrin affinity and clustering plays a key role in the control of cell adhesion and migration. The protein ICAP-1 alpha (integrin cytoplasmic domain-associated protein-1 alpha) binds to the cytoplasmic domain of the beta(1A) integrin and controls cell spreading on fibronectin. Here, we demonstrate that, despite its ability to interact with beta(1A) integrin, ICAP-1 alpha is not recruited in focal adhesions, whereas it is colocalized with the integrin at the ruffling edges of the cells. ICAP-1 alpha induced a rapid disruption of focal adhesions, which may result from the ability of ICAP-1 alpha to inhibit the association of beta(1A) integrin with talin, which is crucial for the assembly of these structures. ICAP-1 alpha-mediated dispersion of beta(1A) integrins is not observed with beta(1D) integrins that do not bind ICAP. This strongly suggests that ICAP-1 alpha action depends on a direct interaction between ICAP-1 alpha and the cytoplasmic domain of the beta(1) chains. Altogether, these results suggest that ICAP-1 alpha plays a key role in cell adhesion by acting as a negative regulator of beta(1) integrin avidity.     

Role of multiple beta1 integrins in cell adhesion to the disintegrin domains of ADAMs 2 and 3

ADAM disintegrin domains can support integrin-mediated cell adhesion. However, the profile of which integrins are employed for adhesion to a given disintegrin domain remains unclear. For example, we suggested that the disintegrin domains of mouse sperm ADAMs 2 and 3 can interact with the alpha6beta1 integrin on mouse eggs. Others concluded that these disintegrin domains interact instead with the alpha9beta1 integrin. To address these differing results, we first studied adhesion of mouse F9 embryonal carcinoma cells and human G361 melanoma cells to the disintegrin domains of mouse ADAMs 2 and 3. Both cell lines express alpha6beta1 and alpha9beta1 integrins at their surfaces. Antibodies to the alpha6 integrin subunit inhibited adhesion of both cell lines. An antibody that recognizes human alpha9 integrin inhibited adhesion of G361 cells. VLO5, a snake disintegrin that antagonizes alpha4beta1 and alpha9beta1 integrins, potently inhibited adhesion of both cell lines. We next explored expression of the alpha9 integrin subunit in mouse eggs. In contrast to our ability to detect alpha6beta1, we were unable to convincingly detect alpha9beta1 integrin on the surface of mouse eggs. Moreover, treatment of mouse eggs with 250 nm VLO5, which is 250 fold over its approximately IC(50) for inhibition of somatic cell adhesion, had minimal effect on sperm-egg binding or fusion. We did detect alpha9 integrin protein on epithelial cells of the oviduct. Additional studies showed that antibodies to the alpha6 and alpha7 integrins additively inhibited adhesion of mouse trophoblast stem cells and that an antibody to the alpha4 integrin inhibited adhesion of MOLT-3 cells to these disintegrin domains: Our data suggest that multiple integrins (on the same cell) can participate in adhesion to a given ADAM disintegrin domain and that interactions between ADAMs and integrins may be important for sperm transit through the oviduct.     

The integrin alpha 7 cytoplasmic domain regulates cell migration, lamellipodia formation, and p130CAS/Crk coupling

The integrin alpha(7)beta(1) is the major laminin-binding integrin in skeletal, heart, and smooth muscle and is a receptor for laminin-1 and -2. It mediates myoblast migration on laminin-1 and -2 and thus might be involved in muscle development and repair. Previously we have shown that alpha(7)B as well as the alpha(7)A and -C splice variants induce cell motility on laminin when transfected into nonmotile HEK293 cells. In this study we have investigated the role of the cytoplasmic domain of alpha(7) in the laminin-induced signal transduction of alpha(7)beta(1) integrin regulating cell adhesion and migration. Deletion of the cytoplasmic domain did not affect assembly of the mutated alpha(7)Deltacyt/beta(1) heterodimer on the cell surface or adhesion of alpha(7)Deltacyt-transfected cells to laminin. The motility of these cells on the laminin-1/E8 fragment, however, was significantly reduced to the level of mock-transfected cells; lamellipodia formation and polarization of the cells were also impaired. Adhesion to the laminin-1/E8 fragment induced tyrosine phosphorylation of the focal adhesion kinase, paxillin, and p130(CAS) as well as the formation of a p130(CAS)-Crk complex in wild-type alpha(7)B-transfected cells. In alpha(7)BDeltacyt cells, however, the extent of p130(CAS) tyrosine formation was reduced and formation of the p130(CAS)-Crk complex was impaired, with unaltered levels of p130(CAS) and Crk protein levels. These findings indicate adhesion-dependent regulation of p130(CAS)/Crk complex formation by the cytoplasmic domain of alpha(7)B integrin after cell adhesion to laminin-1/E8 and imply alpha(7)B-controlled lamellipodia formation and cell migration through the p130(CAS)/Crk protein complex.     

L1CAM: A major driver for tumor cell invasion and motility

The L1 cell adhesion molecule (L1CAM) plays a major role in the development of the nervous system and in the malignancy of human tumors. In terms of biological function, L1CAM comes along in two different flavors: (1) a static function as a cell adhesion molecule that acts as a glue between cells; (2) a motility promoting function that drives cell migration during neural development and supports metastasis of human cancers. Important factors that contribute to the switch in the functional mode of L1CAM are: (1) the cleavage from the cell surface by membrane proximal proteolysis and (2) the ability to change binding partners and engage in L1CAM-integrin binding. Recent studies have shown that the cleavage of L1CAM by metalloproteinases and the binding of L1CAM to integrins via its RGD-motif in the sixth Ig-domain activate signaling pathways distinct from the ones elicited by homophilic binding. Here we highlight important features of L1CAM proteolysis and the signaling of L1CAM via integrin engagement. The novel insights into L1CAM downstream signaling and its regulation during tumor progression and epithelial-mesenchymal transition (EMT) will lead to a better understanding of the dualistic role of L1CAM as a cell adhesion and/or motility promoting cell surface molecule.