Distinct ligand-binding modes for integrin alpha(v)beta(3)-mediated adhesion to fibronectin versus vitronectin.

Cell surface integrins can adopt distinct conformations in response to ligand binding and intracellular signals. Several integrins including alpha(v)beta(3) can bind to multiple ligands. The binding of alpha(v)beta(3) to fibronectin and vitronectin was used as a model to determine whether the same or distinct forms of the receptor were utilized in strong binding to the two different ligands. A spinning-disc device was used to measure the relative strength of the alpha(v)beta(3)-ligand bonds. The initial binding reaction for both ligands occurred in the absence of metabolic energy and resulted in a strong adhesion to fibronectin but a weak adhesion to vitronectin. Increases in the strength of the alpha(v)beta(3)-vitronectin bond required phosphorylation of the beta(3) cytoplasmic domain, intracellular signals, and the binding of cytoskeletal proteins to cytoplasmic domains of beta(3) controlled by Tyr-747 and Tyr-759. In contrast, alpha(v)beta(3)-mediated adhesion to fibronectin was unaffected by phorbol 12-myristate 13-acetate, mutations of Tyr-747 and Tyr-759 to phenylalanine, or availability of metabolic energy. This suggests that strong adhesion to fibronectin used the initial binding conformation, whereas strong binding to vitronectin required signaling-induced changes in the conformation of alpha(v)beta(3).     

Cytokines modulate integrin alpha(v)beta(3)-mediated human endothelial cell adhesion and calcium signaling.

Angiogenesis is a complex process regulated by the interactions of endothelial cells with cytokines and the adhesive protein matrix. The cytokines basic fibroblast growth factor (bFGF) and tumor necrosis factor-alpha (TNF-alpha) are two of the modulators of angiogenesis. One mechanism by which these cytokines induce their effects may be through the regulation of integrin adhesion receptor activity, in particular, alpha(v)beta(3). In this study, we examined the ability of these angiogenic factors to modulate the adhesion of human umbilical vein endothelial cells (HUVECs) to immobilized disintegrins (i.e., rhodostomin and arietin), which are specific in antagonizing integrin alpha(v)beta(3) in cells. As these disintegrins were immobilized as substrates, they acted as agonists to induce HUVEC adhesion in a dose- and alpha(v)beta(3)-dependent manner. In addition, adhesion also triggered a sustained increase of intracellular free calcium. Furthermore, bFGF-primed HUVECs potentiated, but TNF-alpha primed cells attenuated, about 50% adhesion events and calcium signaling triggered by immobilized disintegrin compared to naive cells, respectively. The mechanisms of modulating alpha(v)beta(3)-dependent HUVEC adhesion by cytokines may be related to changes of integrin alpha(v)beta(3) conformation, as demonstrating the antagonistic effect of Mn(2+) on decreased adhesion by TNF-alpha pretreatment, and confirmed with flow cytometric analysis probed by anti-LIBS1 mAb. However, cytokine pretreatment did not alter the expression of this integrin on the cell surface, as determined by flow cytometry. Phosphoinositide-3 kinase may be one of the signaling molecules involved in the enhanced adhesion of bFGF-primed cells.     

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.     

De novo expression of the integrin alpha5beta1 regulates alphavbeta3-mediated adhesion and migration on fibrinogen

Recent evidence demonstrates that interactions between different integrins that are present on the cell surface can strongly influence the adhesive function of individual receptors. In this report, we show that Chinese hamster ovary cells that express the integrin alphavbeta3 in the absence of alpha5beta1 demonstrate increased adhesion and migration on fibrinogen. Furthermore, alphavbeta3-mediated adhesion to fibrinogen is not augmented by the soluble agonist, MnCl2, suggesting that alphavbeta3 exists in a higher affinity state in these cells. De novo expression of wild-type alpha5beta1 negatively regulates alphavbeta3-mediated adhesion and migration. This effect is not seen with expression of a chimeric alpha5beta1 integrin in which the cytoplasmic portion of the alpha5 integrin subunit is replaced by the cytoplasmic portion of the alpha4 integrin. In addition, it does not require ligation of alpha5beta1 by fibronectin. Cells that express a constitutively active beta3 integrin that contains a point mutation in the conserved membrane proximal region of the cytoplasmic tail, D723R, are resistant to the effect of alpha5beta1 expression. These data provide additional evidence of "cross-talk" between the integrins alpha5beta1 and alphavbeta3, and support the idea that alpha5beta1 regulates alphavbeta3-mediated ligand binding. This provides a relevant biological mechanism whereby variations in alpha5beta1 expression in vivo may modulate activation of alphavbeta3 to influence its adhesive function.     

Specific binding of integrin alpha v beta 3 to the fibrinogen gamma and alpha E chain C-terminal domains

Integrin alpha v beta 3, a widely distributed fibrinogen receptor, recognizes the RGD572-574 motif in the alpha chain of human fibrinogen. However, this motif is not conserved in other species, nor is it required for alpha v beta 3-mediated fibrin clot retraction, suggesting that fibrinogen may have other alpha v beta 3 binding sites. Fibrinogen has conserved C-terminal domains in its alpha (E variant), beta, and gamma chains (designated alpha EC, beta C, and gamma C, respectively), but their function in cell adhesion is not known, except that alpha IIb beta 3, a platelet fibrinogen receptor, binds to the gamma C HHLGGAKQAGDV400-411 sequence. Here we used mammalian cells expressing recombinant alpha v beta 3 to show that recombinant alpha EC and gamma C domains expressed in bacteria specifically bind to alpha v beta 3. Interaction between alpha v beta 3 and gamma C or alpha EC is blocked by LM609, a function-blocking anti-alpha v beta 3 mAb, and by RGD peptides. alpha v beta 3 does not require the HHLGGAKQAGDV400-411 sequence of gamma C for binding, and alpha EC does not have such a sequence, indicating that the alpha v beta 3 binding sites are distinct from those of alpha IIb beta 3. A small fragment of gamma C (residues 148-226) supports alpha v beta 3 adhesion, suggesting that an alpha v beta 3 binding site is located within the gamma chain 148-226 region. We have reported that the CYDMKTTC sequence of beta 3 is responsible for the ligand specificity of alpha v beta 3. gamma C and alpha EC do not bind to wild-type alpha v beta 1, but do bind to the alpha v beta 1 mutant (alpha v beta 1-3-1), in which the CYDMKTTC sequence of beta 3 is substituted for the corresponding beta 1 sequence CTSEQNC. This suggests that gamma C and alpha EC contain determinants for fibrinogen's specificity to alpha v beta 3. These results suggest that fibrinogen has potentially significant novel alpha v beta 3 binding sites in gamma C and alpha EC.     

P21-activated kinase 4 interacts with integrin alpha v beta 5 and regulates alpha v beta 5-mediated cell migration

p21-activated kinase 1 (PAK1) can affect cell migration (Price et al., 1998; del Pozo et al., 2000) and modulate myosin light chain kinase and LIM kinase, which are components of the cellular motility machinery (Edwards, D.C., L.C. Sanders, G.M. Bokoch, and G.N. Gill. 1999. Nature Cell Biol. 1:253-259; Sanders, L.C., F. Matsumura, G.M. Bokoch, and P. de Lanerolle. 1999. SCIENCE: 283:2083-2085). We here present a novel cell motility pathway by demonstrating that PAK4 directly interacts with an integrin intracellular domain and regulates carcinoma cell motility in an integrin-specific manner. Yeast two-hybrid screening identified PAK4 binding to the cytoplasmic domain of the integrin beta 5 subunit, an association that was also found in mammalian cells between endogenous PAK4 and integrin alpha v beta 5. Furthermore, we mapped the PAK4 binding to the membrane-proximal region of integrin beta 5, and identified an integrin-binding domain at aa 505-530 in the COOH terminus of PAK4. Importantly, engagement of integrin alpha v beta 5 by cell attachment to vitronectin led to a redistribution of PAK4 from the cytosol to dynamic lamellipodial structures where PAK4 colocalized with integrin alpha v beta 5. Functionally, PAK4 induced integrin alpha v beta 5-mediated, but not beta1-mediated, human breast carcinoma cell migration, while no changes in integrin cell surface expression levels were observed. In conclusion, our results demonstrate that PAK4 interacts with integrin alpha v beta 5 and selectively promotes integrin alpha v beta 5-mediated cell migration.     

Involvement of integrin alpha(v)beta(3) and cell adhesion molecule L1 in transendothelial migration of melanoma cells

Tumor metastasis involves many stage-specific adhesive interactions. The expression of several cell adhesion molecules, notably the integrin alpha(v)beta(3), has been associated with the metastatic potential of tumor cells. In this study, we used a novel in vitro assay to examine the role of alpha(v)beta(3) in the transmigration of melanoma cells through a monolayer of human lung microvascular endothelial cells. Confocal microscopy revealed the presence of the integrin alpha(v)beta(3) on melanoma membrane protrusions and pseudopods penetrating the endothelial junction. alpha(v)beta(3) was also enriched in heterotypic contacts between endothelial cells and melanoma cells. Transendothelial migration of melanoma cells was inhibited by either a cyclic Arg-Gly-Asp peptide or the anti-alpha(v)beta(3) monoclonal antibody LM609. Although both platelet endothelial cell adhesion molecule-1 and L1 are known to bind integrin alpha(v)beta(3), only L1 serves as a potential ligand for alpha(v)beta(3) during melanoma transendothelial migration. Also, polyclonal antibodies against L1 partially inhibited the transendothelial migration of melanoma cells. However, addition of both L1 and alpha(v)beta(3) antibodies did not show additive effects, suggesting that they are components of the same adhesion system. Together, the data suggest that interactions between the integrin alpha(v)beta(3) on melanoma cells and L1 on endothelial cells play an important role in the transendothelial migration of melanoma cells.     

Characterisation of alpha3beta1 and alpha(v)beta3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of beta1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, alpha3beta1 and alpha(v)beta3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with beta1,6-branches and short polylactosamine chains. In WM9 cells, alpha3beta1 integrin was more variously glycosylated than alpha(v)beta3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and alpha3beta1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of alpha(v)beta3 integrin glycans in melanoma or in any cancer cells.     

The integrin complex alpha v beta 3 participates in the adhesion of microvascular endothelial cells to fibronectin.

Fibronectin is a major adhesive glycoprotein of the vascular basement membrane. Since fibronectin is also found in the interstitium, it may be important not only for attachment but also for endothelial cell migration during neovascularization. We have analyzed how human dermal microvascular endothelial cells use their diverse set of integrin receptors to interact with this ligand. Immunofluorescent staining with specific antibodies identified both beta 1 and beta 3 integrin receptor complexes in focal adhesion plaques on cells adhering to immobilized fibronectin. Adhesion assays with blocking monoclonal antibodies implicated both beta 1 and beta 3 complexes, specifically alpha 5 beta 1 and alpha v beta 3, in the initial adhesion of cells to fibronectin. Finally, ligand affinity chromatography of extracts of surface radiolabeled cells established that both alpha 5 beta 1 and alpha v beta 3 could bind to the 110-kDa cell-binding fragment of fibronectin. An additional receptor complex composed of an alpha v subunit and a beta 5-like subunit was also detected. These results provide evidence that microvascular endothelial cells use multiple integrin receptors, from several beta families, to attach to fibronectin surfaces.     

Arginine-glycine-aspartic acid binding leading to molecular stabilization between integrin alpha v beta 3 and its ligand.

Cell adhesion is characterized by an integrin-mediated ligand binding event followed by reorganization of the actin-cytoskeleton leading to cell spreading and/or migration. In this report we examine the role of integrin alpha v beta 3 in mediating cell attachment to vitronectin or a RGD-containing peptide in the presence of cytochalasin B to prevent actin polymerization. Under these conditions cell attachment to a RGD-containing peptide can be dissociated by excess soluble ligand whereas cells attached to vitronectin cannot. These results suggest that alpha v beta 3-mediated cell attachment to vitronectin results in a highly stabilized interaction that is independent of the actin-cytoskeleton. To investigate the molecular nature of this interaction alpha v beta 3 was purified to homogeneity, and its binding properties toward various ligands were measured in a solid-phase receptor assay. The data indicate that alpha v beta 3 binds to vitronectin or fibronectin in a nondissociable manner whereas a RGD-containing peptide derived from vitronectin binds specifically but is completely dissociable with a Kd of 9.4 x 10(-7) M. Moreover, chemical modification of alpha v beta 3 with limited glutaraldehyde treatment allowed vitronectin to bind in a RGD-dependent and dissociable manner, suggesting that receptor conformational changes or specific amino acid residues proximal to the ligand binding site(s) are involved in the stabilization event. Thus, in the absence of cytoskeletal proteins or other cellular components, integrin alpha v beta 3-ligand binding involves recognition of the RGD sequence leading to a highly stabilized protein-protein association.     

L-plastin peptide activation of alpha(v)beta(3)-mediated adhesion requires integrin conformational change and actin filament disassembly

L-plastin (LPL) is a leukocyte actin binding protein previously implicated in the activation of the integrin alpha(M)beta(2) on polymorphonuclear neutrophils. To determine the role for LPL in integrin activation, K562 cell adhesion to vitronectin via alpha(v)beta(3), a well-studied model for activable integrins, was examined. Cell permeant versions of peptides based on the N-terminal sequence of LPL and the LPL headpiece domain both activated alpha(v)beta(3)-mediated adhesion. In contrast to adhesion induced by treatment with phorbol 12-myristate 13-acetate (PMA), LPL peptide-activated adhesion was independent of integrin beta(3) cytoplasmic domain tyrosines and was not inhibited by cytochalasin D. Also in contrast to PMA, LPL peptides synergized with RGD ligand or Mn(2+) for generation of a conformational change in alpha(v)beta(3) associated with the high affinity state of the integrin, as determined by binding of a ligand-induced binding site antibody. Although LPL and ligand showed synergy for ligand-induced binding site expression when actin depolymerization was inhibited by jasplakinolide, LPL peptide-induced adhesion was inhibited. Thus, both actin depolymerization and ligand-induced integrin conformational change are required for LPL peptide-induced adhesion. We hypothesize that the critical steps of increased integrin diffusion and affinity enhancement may be linked via modulation of the function of the actin binding protein L-plastin.     

Human neural cell adhesion molecule L1 and rat homologue NILE are ligands for integrin alpha v beta 3

Integrin alpha v beta 3 is distinct in its capacity to recognize the sequence Arg-Gly-Asp (RGD) in many extra-cellular matrix (ECM) components. Here, we demonstrate that in addition to the recognition of ECM components, alpha v beta 3 can interact with the neural cell adhesion molecule L1-CAM; a member of the immunoglobulin superfamily (IgSF). M21 melanoma cells displayed significant Ca(++)-dependent adhesion and spreading on immunopurified rat L1 (NILE). This adhesion was found to be dependent on the expression of the alpha v-integrin subunit and could be significantly inhibited by an antibody to the alpha v beta 3 heterodimer. M21 cells also displayed some alpha v beta 3-dependent adhesion and spreading on immunopurified human L1. Ligation between this ligand and alpha v beta 3 was also observed to promote significant haptotactic cell migration. To map the site of alpha v beta 3 ligation we used recombinant L1 fragments comprising the entire extracellular domain of human L1. Significant alpha v beta 3-dependent adhesion and spreading was evident on a L1 fragment containing Ig-like domains 4, 5, and 6. Importantly, mutation of an RGD sequence present in the sixth Ig-like domain of L1 abrogated M21 cell adhesion. We conclude that alpha v beta 3-dependent recognition of human L1 is dependent on ligation of this RGD site. Despite high levels of L1 expression the M21 melanoma cells did not display significant adhesion via a homophilic L1-L1 interaction. These data suggest that M21 melanoma cells recognize and adhere to L1 through a mechanism that is primarily heterophilic and integrin dependent. Finally, we present evidence that melanoma cells can shed and deposit L1 in occluding ECM. In this regard, alpha v beta 3 may recognize L1 in a cell-cell or cell- substrate interaction.     

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.     

Alpha v beta 3 and alpha v beta 5 integrins and their role in muscle precursor cell adhesion

BACKGROUND INFORMATION: Functional adaptation of skeletal muscle is a requirement for different muscle groups (e.g. craniofacial, ocular and limb) to undergo site-specific changes. Such tissue remodelling depends on dynamic interactions between muscle cells and their extracellular matrix, via participation of multifunctional molecules such as integrins. In view of data suggesting a role in fundamental muscle biology and muscle development in other systems, the present study has focused on expression and function of alpha v integrins, in cultured adult human craniofacial muscle (masseter) precursor cells and myotubes, and the predominantly fibroblastic IC (interstitial cells) population. RESULTS AND CONCLUSIONS: Flow-cytometric phenotyping and immunofluorescence phenotyping show that alpha v, alpha v beta 3 and alpha v beta 5 are expressed in all mononuclear cells (muscle precursors and IC) seeded on muscle extracellular molecules such as gelatin, VN (vitronectin) and FN (fibronectin). In this system, blockade of alpha v activity using a function-perturbing antibody abrogates cell migration on VN and FN. alpha v integrins act predominantly as VN receptors as cell-substrate attachment is diminished when alpha v neutralizing agents are introduced into cultures seeded on VN, and this inhibition is reversible; these integrins also appear to be minor FN receptors. These results demonstrate that the alpha v subset of integrins present on both myogenic precursors and IC is an essential cohort of VN and, to a lesser extent, FN receptors mediating cell adhesion and, either directly or indirectly, arbiters of cell motility.     

Cooperation between thrombospondin-1 type 1 repeat peptides and alpha(v)beta(3) integrin ligands to promote melanoma cell spreading and focal adhesion kinase phosphorylation.

CD47-binding sequences from the carboxyl-terminal domain of thrombospondin-1 (TSP1) are known to regulate activity of the alpha(v)beta(3) integrin (Gao, G., Lindberg, F. P., Dimitry, J. M., Brown, E. J., and Frazier, W. A. (1996) J. Cell Biol. 135, 533-544). Here we show that peptides from the type 1 repeats of TSP1 also stimulate alpha(v)beta(3) integrin function in melanoma cells. Addition of soluble peptide 246 (KRFKQDGGWSHWSPWSS) enhances spreading of A2058 melanoma cells on several alpha(v)beta(3) integrin ligands, including vitronectin, recombinant TSP1 fragments containing the Arg-Gly-Asp sequence, and native TSP1. This activity requires the Trp residues and is independent of CD36-binding sequences in the type 1 repeats. Recombinant type 1 repeats expressed as a glutathione S-transferase fusion protein also enhance spreading on vitronectin and TSP1. Activation of alpha(v)beta(3) integrin by the soluble peptide 246 stimulates organization of F-actin and increases tyrosine phosphorylation of focal adhesion kinase. In contrast, direct adhesion of melanoma cells on immobilized peptide 246 inhibits tyrosine phosphorylation of focal adhesion kinase. Stimulation of alpha(v)beta(3) integrin function by the type 1 repeat peptide differs from that induced by CD47-binding TSP1 peptides in that heparan sulfate proteoglycans are required and pertussis toxin does not inhibit the former activity. Thus, the type 1 repeats contain a second sequence of TSP1 that can enhance alpha(v)beta(3) integrin signaling, and these two sequences stimulate recognition of both vitronectin and TSP1 by the alpha(v)beta(3) integrin.     

alpha v beta3 and alpha5beta1 integrin recycling pathways dictate downstream Rho kinase signaling to regulate persistent cell migration

Accumulating evidence suggests that integrin recycling regulates cell migration. However, the lack of reagents to selectively target the trafficking of individual heterodimers, as opposed to endocytic transport as a whole, has made it difficult to define the contribution made by particular recycling pathways to directional cell movement. We show that autophosphorylation of protein kinase D1 (PKD1) at Ser(916) is necessary for its association with alphavbeta3 integrin. Expression of PKD1(916A) or the use of mutants of beta3 that do not bind to PKD1 selectively inhibits short-loop, Rab4-dependent recycling of alphavbeta3, and this suppresses the persistence of fibroblast migration. However, we report that short-loop recycling does not directly contribute to fibroblast migration by moving alphavbeta3 to the cell front, but by antagonizing alpha5beta1 recycling, which, in turn, influences the cell's decision to migrate with persistence or to move randomly.     

Purification and functional characterization of integrin alpha v beta 5. An adhesion receptor for vitronectin.

We have purified a novel member of the integrin gene family from placenta that serves as a vitronectin receptor. This integrin is composed of the alpha v subunit and a beta subunit that we designate beta 5. Purification was accomplished by immunodepleting a placental extract of integrin alpha v beta 3, allowing us to purify alpha v beta 5 from the remaining extract by monoclonal antibody affinity chromatography on LM 142-Sepharose, which binds to the alpha v subunit. Purification to homogeneity was subsequently achieved by affinity chromatography on wheat germ lectin-Sepharose. Western blot analysis with antibodies raised against alpha v beta 5 and alpha v beta 3 demonstrated that beta 3 and beta 5 were distinct but confirmed that the alpha subunit of the two integrins were immunologically identical. Similarly, antibodies that bind beta 3 proximal to the ligand-binding site failed to react with beta 5, indicating an architectural difference at the ligand-binding site of these related integrins. This structural difference apparently results in a functional distinction, since purified alpha v beta 3 bound to vitronectin, fibrinogen, von Willebrand factor, and fibronectin, whereas integrin alpha v beta 5 bound preferentially to vitronectin. Finally, we demonstrate by three criteria that beta 5 and beta x, the latter of which was identified in lung carcinoma cells (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), are identical. First, peptide maps of beta x and beta 5 are identical. Secondly, polyclonal antibodies raised against alpha v beta 5 immunoprecipitate both beta 5 and beta x, and finally, the amino-terminal amino acid sequences of beta x and beta 5 are identical.     

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.