The integrin beta 1 subunit associates with the vitronectin receptor alpha v subunit to form a novel vitronectin receptor in a human embryonic kidney cell line.

We describe a novel integrin heterodimer on the surface of the human embryonic kidney cell line 293. This receptor is comprised of alpha v and beta 1 subunits, each of which has been previously found in association with other integrin subunits. This alpha v.beta 1 complex was identified as the predominant vitronectin receptor (VnR) on the surface of 293 cells by immunoprecipitation with antibodies raised against the alpha v subunit. Polymerase chain reaction analysis detected mRNAs for alpha v and beta 1 subunits while no evidence was obtained for beta 2, beta 3, or alpha IIb integrin subunit mRNA. Immunoprecipitation of surface-iodinated proteins with antibodies to alpha v gave bands of 150 and 120 kDa. The 120-kDa band reacted with antibodies to beta 1 in immunoblotting experiments. 293 cells adhere to vitronectin, fibronectin, laminin, and collagen IV, while von Willebrand factor and fibrinogen, known ligands of the VnR (alpha v.beta 3), did not support adhesion. A polyclonal antibody directed against both subunits of the VnR (alpha v, beta 3) inhibits attachment of 293 cells to vitronectin but not to other adhesive proteins. A beta 1-specific monoclonal inhibited attachment to fibronectin, laminin, and collagen IV, known ligands of beta 1 integrins, as well as vitronectin. This novel (alpha v. beta 1) VnR thus appears to mediate cell adhesion exclusively to vitronectin, in contrast to previously described VnRs which have multiple ligands.     

Puromycin insensitive leucyl-specific aminopeptidase (PILSAP) is involved in the activation of endothelial integrins

We previously reported that mouse orthologue of puromycin insensitive leucyl-specific aminopeptidase (mPILSAP) played an important role in angiogenesis by regulating the proliferation and migration of endothelial cells (ECs) (Miyashita et al., 2002. Blood 99:3241-3249). Here, we examined the mechanism as to how mPILSAP regulates the migration of ECs. Cell adhesion through integrins plays a crucial role in cell migration, and ECs use at least type-1 collagen receptor integrin alpha2beta1, fibronectin receptor alpha5beta1, and vitronectin receptors alphavbeta3 and alphavbeta5. mPILSAP antisense oligodeoxynucleotide (AS-ODN) or leucinethiol (LT), a leucyl-aminopeptidase inhibitor, did not affect the attachment but did significantly inhibit the spreading of cells of the murine endothelial cell line MSS31 when they were plated on vitronectin-, fibronectin-, or type-1 collagen, although they did not affect the expression of integrin alpha2, alpha5, alphav, beta1, beta3, and beta5 subunits on the cell surface. AS-ODN and LT also inhibited the tyrosine phosphorylation of FAK when cells were plated on vitronectin, fibronectin, or type-1 collagen. This inhibition of cell spreading and of tyrosine phosphorylation of FAK could be negated by Mg(2+). These results suggest that mPILSAP is involved in the activation of endothelial integrins.     

Regulation of integrin alpha vbeta 3-mediated endothelial cell migration and angiogenesis by integrin alpha5beta1 and protein kinase A

Recent studies indicate that angiogenesis depends, in part, on ligation of integrin alpha(5)beta(1) by fibronectin. Evidence is now provided that integrin alpha(5)beta(1) regulates the function of integrin alpha(v)beta(3) on endothelial cells during their migration in vitro or angiogenesis in vivo. Secretion of fibronectin by endothelial cells leads to the ligation of integrin alpha(5)beta(1), which potentiates alpha(v)beta(3)-mediated migration on vitronectin without influencing alpha(v)beta(3)-mediated cell adhesion. Endothelial cell attachment to vitronectin suppresses protein kinase A (PKA) activity, while addition of soluble anti-alpha(5)beta(1) restores this activity. Moreover, agents that activate intracellular PKA, such as forskolin, dibutyryl cAMP or alpha(5)beta(1) antagonists, suppress endothelial cell migration on vitronectin in vitro or angiogenesis in vivo. In contrast, inhibitors of PKA reverse the anti-migratory or anti-angiogenic effects mediated by alpha(5)beta(1) antagonists. Therefore, alpha(v)beta(3)-mediated endothelial cell migration and angiogenesis can be regulated by PKA activity, which depends on the ligation state of integrin alpha(5)beta(1).     

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes

We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.     

Transforming growth factor-beta 1 modulates beta 1 and beta 5 integrin receptors and induces the de novo expression of the alpha v beta 6 heterodimer in normal human keratinocytes: implications for wound healing

The molecular mechanism underlying the promotion of wound healing by TGF-beta 1 is incompletely understood. We report that TGF-beta 1 regulates the regenerative/migratory phenotype of normal human keratinocytes by modulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratified cultured epidermis, TGF-beta 1: (a) strongly upregulates the expression of the fibronectin receptor alpha 5 beta 1, the vitronectin receptor alpha v beta 5, and the collagen receptor alpha 2 beta 1 by differentially modulating the synthesis of their alpha and beta subunits; (b) downregulates the multifunctional alpha 3 beta 1 heterodimer; (c) induces the de novo expression and surface exposure of the alpha v beta 6 fibronectin receptor; (d) stimulates keratinocyte migration toward fibronectin and vitronectin; (e) induces a marked perturbation of the general mechanism of polarized domain sorting of both beta 1 and beta 4 dimers; and (f) causes a pericellular redistribution of alpha v beta 5. These data suggest that alpha 5 beta 1, alpha v beta 6, and alpha v beta 5, not routinely used by keratinocytes resting on an intact basement membrane, act as "emergency" receptors, and uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human wounds. Indeed, TGF-beta 1 reproduces the integrin expression pattern of keratinocytes located at the injury site, particularly of cells in the migrating epithelial tongue at the leading edge of the wound. Since these keratinocytes are inhibited in their proliferative capacity, these data might account for the apparent paradox of a TGF-beta 1-dependent stimulation of epidermal wound healing associated with a growth inhibitory effect on epithelial cells.     

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.     

Trypsin stimulates integrin alpha(5)beta(1)-dependent adhesion to fibronectin and proliferation of human gastric carcinoma cells through activation of proteinase-activated receptor-2

Trypsin is widely expressed in various non-pancreatic tissues at low levels and overexpressed in some types of human cancers. In the present study, we found that trypsin stimulates integrin-dependent adhesion and growth of MKN-1 human gastric carcinoma cells. MKN-1 cells expressed both proteinase-activated receptor-1 (PAR-1) and PAR-2, which are activated by thrombin and trypsin, respectively. Both trypsin and the PAR-2 ligand SLIGKV promoted integrin alpha(5)beta(1)-mediated adhesion of MKN-1 cells to fibronectin, and less effectively integrin alpha(v)beta(3)-mediated cell adhesion to vitronectin, but not that to type IV collagen or laminin-1 at all. Thrombin and the PAR-1 ligand SFLLRN promoted the cell adhesion to vitronectin more strongly than trypsin or the PAR-2 ligand, but not the cell adhesion to fibronectin at all. The cell adhesion-stimulating effect of the PAR-2 ligand was significantly reduced by the pre-treatment of cells with trypsin, indicating that the effect of trypsin is mediated by PAR-2 activation. The trypsin-stimulated cell adhesion to vitronectin, but not to fibronectin, was effectively inhibited by the G(i) protein blocker pertussis toxin, and both cell adhesions were completely inhibited by the Src kinase inhibitor herbimycin A. Furthermore, trypsin and the PAR-2 ligand stimulated growth of MKN-1 cells more strongly than thrombin or the PAR-1 ligand. These results show that trypsin regulates cellular adhesion and proliferation by inducing PAR-2/G protein signalings, and that the integrin alpha(5)beta(1)- and integrin alpha(v)beta(3)-dependent cell adhesions are regulated by different PAR/G protein signalings.     

Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein.

Integrins are a complex family of divalent cation-dependent cell adhesion receptors composed of one alpha and one beta subunit noncovalently bound to one another. A subset of integrins contains the alpha v subunit in association with one of several beta subunits (e.g. beta 3, beta 5, beta 1). We have recently identified a novel integrin beta subunit, beta 6, that is present in a number of epithelial cell lines. Using a polyclonal antibody raised against the carboxyl-terminal peptide of beta 6, we have now identified the integrin heterodimer, alpha v beta 6, on the surface of two human carcinoma cell lines. Using affinity chromatography of lysates from the pancreatic carcinoma cell line, FG-2, we demonstrate that alpha v beta 6 binds to fibronectin, but not to vitronectin or collagen I. In contrast, the alpha v beta 5 integrin, which is also expressed on FG-2 cells, binds exclusively to vitronectin. Immobilized collagen I does not interact with alpha v integrins, but binds beta 1-containing integrins. Both alpha v beta 6 and alpha v beta 5 are eluted from their respective immobilized ligands by a hexa-peptide containing the sequence Arg-Gly-Asp (RGD). RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+. These results suggest that alpha v beta 6 functions as an RGD-dependent fibronectin receptor in FG-2 carcinoma cells. In agreement with this notion, cell adhesion assays show that FG-2 cell attachment to fibronectin is only partially inhibited by anti-beta 1 integrin antibodies, implying that other fibronectin receptors may be involved. Taken together with recent reports on the vitronectin receptor function of alpha v beta 5, our results suggest that the previously described carcinoma cell integrin, alpha v beta x (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), is a mixture of at least two different receptors: alpha v beta 5, mediating adhesion to vitronectin, and alpha v beta 6, mediating adhesion to fibronectin.     

Matrix metalloproteinase-2 is involved in A549 cell migration on laminin-10/11

We have reported that laminin-10/11 strongly promotes migration of A549 human lung carcinoma cells by activating the alpha3beta1 integrin-dependent signaling pathway. To elucidate the mechanism involved, we investigated whether matrix metalloproteinases (MMPs) are involved in cell migration on laminin-10/11. Here, we demonstrate that laminin-10/11, but not fibronectin which does not greatly promote A549 cell movement, stimulated MMP-2 secretion approximately 3-fold. The cell migration-promoting activity of laminin-10/11 was down-regulated by an MMP inhibitor. In addition, cell motility was significantly increased when cells adhered to a mixture of fibronectin and laminin-10/11 with a concomitant decrease of focal contacts, compared with those adhering to fibronectin alone. The enhanced cell migration was partially suppressed by the MMP inhibitor. Furthermore, an anti-alpha3 integrin, but not an anti-alpha5 integrin, antibody induced the activated form of MMP-2. These data suggest that MMP-2 may play an important role in A549 cell migration on laminin-10/11 through an alpha3beta1 integrin-dependent pathway.     

The role of the tetraspanin CD151 in primary keratinocyte and fibroblast functions: implications for wound healing

Previous studies showed that CD151-null mice have a skin wound healing deficit. To gain an understanding of the role of CD151 in re-epithelialisation and dermal contraction, keratinocyte and fibroblast functions were assayed. Primary CD151-null keratinocytes displayed defective migration on Matrigel (a basement membrane equivalent) and laminin-332, the primary adhesion component of basement membranes, but not on collagen-I. Adhesion, spreading and proliferation were also deficient on laminin-332, but not collagen-I. The data suggest that loss of CD151 impairs the function of its primary interaction partners, integrin alpha3beta1- and/or alpha6beta4 which bind to laminin-332. Skin fibroblasts also produce CD151 mRNA. CD151-null fibroblasts migrated significantly faster on collagen I than wild type fibroblasts, confirming that they possess functional collagen receptors. However, no significant decrease in the ability of CD151-null fibroblasts to cause contraction in floating collagen gel assays in response to transforming growth factor beta-1 (TGF-beta1) or platelet derived growth factor (PDGF-BB) was observed, nor was there an effect on fibroblast adhesion or proliferation on collagen-I. The data implicate CD151 as a facilitator of laminin-332-mediated keratinocyte functions that impact on the re-epithelialisation process intrinsic to wound healing and further suggest a potential novel role for CD151 in fibroblast migration.     

Isolation of a novel integrin receptor mediating Arg-Gly-Asp-directed cell adhesion to fibronectin and type I collagen from human neuroblastoma cells. Association of a novel beta 1-related subunit with alpha v

We report the isolation from two human neuroblastoma cell lines of an Arg-Gly-Asp-dependent integrin complex capable of binding to vitronectin, fibronectin, and type I collagen. The two neuroblastoma cell lines, SK-N-SH and IMR-32, exhibit specific attachment to fibronectin and type I collagen. SK-N-SH cells exhibit a much stronger attachment to vitronectin than the IMR-32 cells, which attach poorly to this substrate. Affinity chromatography of octylglucoside extracts of 125I surface-labeled cells on GRGDSPK-Sepharose columns resulted in the specific binding and elution with GRGDSP of three radiolabeled polypeptides with relative molecular masses of 135, 115, and 90 kD when analyzed by SDS-PAGE under nonreducing conditions. In the SK-N-SH cells the 135- and 90-kD polypeptides were more abundant whereas in the IMR-32 cells the 135- and 115-kD polypeptides were more highly expressed. Liposomes prepared from fractions containing all three polypeptides bound to vitronectin, fibronectin, and type I collagen, whereas liposomes prepared from the 135- and 115-kD polypeptides bound only to fibronectin and type I collagen. Polyclonal antibodies against the alpha/beta complexes of both the vitronectin receptor and the fibronectin receptor immunoprecipitated all three polypeptides. A monoclonal antibody against beta 1 immunoprecipitated only the 135- and the 115-kD polypeptides, whereas a monoclonal antibody against beta 3 subunit immunoprecipitated the 135- and 90-kD polypeptides. Although, the 115-kD polypeptide could be recognized by an anti-beta 1 antibody, a comparison of peptide maps generated by V8 protease digestion of the 115-kD polypeptide and beta 1 subunit immunoprecipitated from GRGDSPK-Sepharose flow-through material indicated that these two polypeptides are distinct. Depletion of the 90-kD polypeptide with an anti-beta 3 monoclonal antibody did not effect the ability of the 115- and 135-kD polypeptides to bind to GRGDSPK-Sepharose. These data indicate that the SK-N-SH and IMR-32 neuroblastoma cells express a novel "beta 1-like" integrin subunit that can associate with alpha v and can bind to RGD. We propose to name this beta 1-like subunit beta n. The data reported here thus demonstrate that in these two cell lines alpha v associates with two beta subunits, beta n and beta 3, forming two heterodimers. The alpha v beta n complex mediates binding to fibronectin and type I collagen, whereas the alpha v beta 3 complex mediates binding to vitronectin.     

Rac regulates integrin-mediated endothelial cell adhesion and migration on laminin-8

Blood vessel formation requires endothelial cell interactions with the extracellular matrix through cell surface receptors, and signaling events that control endothelial cell adhesion, migration, and lumen formation. Laminin-8 (alpha4beta1gamma1) is present in all basement membranes of blood vessels in fetal and adult tissues, but despite its importance in vessel formation, its role in endothelial cell adhesion and migration remains undefined. We examined adhesion and migration of HMEC-1 human microvascular endothelial cells on laminin-8 with an emphasis on the integrin-mediated signaling events, as compared with those on laminin-10/11 and fibronectin. We found that laminin-8 was less potent in HMEC-1 cell adhesion than laminin-1, laminin-10/11, and fibronectin, and mediated cell adhesion through alpha6beta1 integrin. Despite its weak cell-adhesive activity, laminin-8 was as potent as laminin-10/11 in promoting cell migration. Cells adhering to laminin-8 displayed streaks of thin actin filaments and formed lamellipodia at the leading edge of the cells, as observed with cells adhering to laminin-10/11, while cells on fibronectin showed thick actin stress fibers and large focal adhesions. Pull-down assays of GTP-loaded Rho, Rac, and Cdc42 demonstrated that Rac, but not Rho or Cdc42, was preferentially activated on laminin-8 and laminin-10/11, when compared with fibronectin. Furthermore, a dominant-negative mutant of Rac suppressed cell spreading, lamellipodial formation, and migration on laminin-8, but not on fibronectin. These results, taken together, indicate that Rac is activated during endothelial cell adhesion to laminin-8, and is pivotal for alpha6beta1 integrin-mediated cell spreading and migration on laminin-8.     

Differential regulation of alpha6beta4 integrin by PKC isoforms in murine skin keratinocytes

In mammalian epidermis, alpha6beta4 integrin is expressed exclusively on the basal layer localized to the hemidesmosomes, where it interacts extracellularly with the laminin-5 ligand. During differentiation, loss of alpha6beta4 is associated with keratinocyte detachment from the basement membrane and upward migration. The protein kinase C (PKC) family of isoforms participates in regulation of integrin function and is linked to skin differentiation. Exposure of primary murine keratinocytes to PKC activators specifically downregulates alpha6beta4 expression. Utilizing recombinant adenoviruses, we selectively overexpressed skin PKC isoforms in primary keratinocytes. PKCdelta and PKCzeta induced downregulation of alpha6beta4 protein expression, leading to reduced keratinocyte attachment to laminin-5 and enhanced gradual detachment from the underlying matrix. In contrast, PKCalpha upregulated alpha6beta4 protein expression, leading to increased keratinocyte attachment to laminin-5 and to the underlying matrix. Altogether, these results suggest distinct roles for specific PKC isoforms in alpha6beta4 functional regulation during the early stages of skin differentiation.     

The vitronectin receptor alpha v beta 3 binds fibronectin and acts in concert with alpha 5 beta 1 in promoting cellular attachment and spreading on fibronectin

The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily of adhesive protein receptors that mediate a wide spectrum of adhesive cellular interactions, including attachment to vitronectin, von Willebrand factor, fibrinogen, and thrombospondin. We have studied the binding of fibronectin to the purified vitronectin receptor, and the role of this receptor in the attachment of cells to fibronectin. A solid-phase microtiter assay was developed to investigate the binding properties of the vitronectin receptor. Purified alpha v beta 3 bound fibronectin with high affinity in a saturable, divalent cation- dependent manner. Binding was inhibited by soluble vitronectin, by RGD- containing peptides, and by LM609, a monoclonal antibody against the vitronectin receptor known to inhibit the binding of adhesive proteins to alpha v beta 3. Immunoinhibition experiments showed that M21 human melanoma cells, which express the fibronectin receptor, alpha 5 beta 1, as well as alpha v beta 3, used both of these integrins to attach and spread on fibronectin. In support of this finding, M21-L cells, a variant cell line that specifically lacks alpha v beta 3 but expresses alpha v beta 1, attached and spread poorly on fibronectin. In addition, alpha v beta 3 from surface-labeled M21 cells was retained, and selectively eluted by RGDS from a fibronectin affinity column. These results indicate that alpha v beta 3 acts in concert with alpha 5 beta 1 in promoting fibronectin recognition by these cells. We conclude that fibronectin binds to the alpha v beta 3 vitronectin receptor specifically and with high affinity, and that this interaction is biologically relevant in supporting cell adhesion to matrix proteins.     

Beta 8 integrins mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin.

Integrins are major receptors used by cells to interact with extracellular matrices. In this paper, we identify the first ligands for the beta 8 family of integrins, presenting evidence that integrin heterodimers containing the beta 8 subunit mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin. A polyclonal antibody, anti-beta 8-Ex, was prepared to a bacterial fusion protein expressing an extracellular portion of the chicken beta 8 subunit. In nonreducing conditions, this antibody immunoprecipitated from surface-labeled embryonic dorsal root ganglia neurons a M(r) 100 k protein, the expected M(r) of the beta 8 subunit, and putative alpha subunit(s) of M(r) 120 k. Affinity-purified anti-beta 8-Ex strongly inhibited sensory neurite outgrowth on laminin-1, collagen IV, and fibronectin-coated substrata. Binding sites were identified in a heat-resistant domain in laminin-1 and in the carboxyl terminal, 40-kDa fibronectin fragment. On substrates coated with the carboxyl terminal fragment of fibronectin, antibodies to beta 1 and beta 8 were only partially effective alone, but were completely effective in combination, at inhibiting neurite outgrowth. Results thus indicate that the integrin beta 8 subunit in association with one or more alpha subunits forms an important set of extracellular matrix receptors on sensory neurons.     

Mechanism of human keratinocyte migration on fibronectin: unique roles of RGD site and integrins.

The migration of human keratinocytes over the wound bed plays an important role in the re-epithelialization of cutaneous wounds. Fibronectin, a large glycoprotein matrix component that is abundant within cutaneous wound beds, promotes keratinocyte migration. However, the mechanisms by which keratinocytes migrate over fibronectin are unknown. In this study, we sought to identify specific sites within the fibronectin molecule that induce keratinocyte locomotion and to characterize the cell surface receptors involved. The data show that the domain within the fibronectin molecule that induces human keratinocyte migration is the 120 kD cell-binding domain close to the carboxyl terminus. The 40 kD heparin-binding domain near the carboxyl terminus and the 45 kD gelatin-binding domain near the amino terminus did not promote keratinocyte migration. In addition, keratinocyte migration on both fibronectin and the 120 kD cell-binding domain was completely inhibited by the presence of GRGDSP peptide, suggesting that keratinocyte migration on fibronectin is mediated by recognizing the RGD sequence located within the cell-binding domain of fibronectin. Furthermore, keratinocytes were able to migrate directly on immobilized RGD substratum. Cell migration on fibronectin is mediated by the alpha 5 beta 1 integrin since antibodies blocking the alpha 5 and the beta 1 subunits completely inhibited keratinocyte migration on fibronectin. In addition, we demonstrate that human keratinocytes express alpha 5 beta 1 integrin in culture by flow cytometry.     

Receptor tyrosine kinase signaling required for integrin alpha v beta 5- directed cell motility but not adhesion on vitronectin

FG human pancreatic carcinoma cells adhere to vitronectin using integrin alpha v beta 5 yet are unable to migrate on this ligand whereas they readily migrate on collagen in an alpha 2 beta 1-dependent manner. We report here that epidermal growth factor receptor (EGFR) activation leads to de novo alpha v beta 5-dependent FG cell migration on vitronectin. The EGFR specific tyrosine kinase inhibitor tyrphostin 25 selectively prevents EGFR autophosphorylation thereby preventing the EGF-induced FG cell migration response on vitronectin without affecting constitutive migration on collagen. Protein kinase C (PKC) activation also leads to alpha v beta 5-directed motility on vitronectin; however, this is not blocked by tyrosine kinase inhibitors. In this case, PKC activation appears to be associated with and downstream of EGFR signaling since calphostin C, an inhibitor of PKC, blocks FG cell migration on vitronectin induced by either PKC or EGF. These findings represent the first report implicating a receptor tyrosine kinase in a specific integrin mediated cell motility event independent of adhesion.     

Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library

Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin.     

Integrin alpha5beta1 supports the migration of Xenopus cranial neural crest on fibronectin

During early embryonic development, cranial neural crest cells emerge from the developing mid- and hindbrain. While numerous studies have focused on integrin involvement in trunk neural crest cell migration, comparatively little is known about mechanisms of cranial neural crest cell migration. We show that fibronectin, but not laminin, vitronectin, or type I collagen can support cranial neural crest cell migration and segmentation in vitro. These behaviors require both the RGD and "synergy" sites located within the central cell-binding domain of fibronectin. While these two sites are sufficient for cranial neural crest cell migration, we find that the second Heparin-binding domain of fibronectin can provide additional support for cranial neural crest cell migration in vitro. Finally, using a function blocking monoclonal antibody, we show that cranial neural crest cell migration on fibronectin requires the integrin alpha5beta1.