Alpha 4 integrin signaling activates phosphatidylinositol 3-kinase and stimulates T cell adhesion to intercellular adhesion molecule-1 to a similar extent as CD3, but induces a distinct rearrangement of the actin cytoskeleton.

Dynamic regulation of beta(2) integrin-dependent adhesion is critical for a wide array of T cell functions. We previously showed that binding of high-affinity alpha(4)beta(1) integrins to VCAM-1 strengthens alpha(L)beta(2) integrin-mediated adhesion to ICAM-1. In this study, we compared beta(2) integrin-mediated adhesion of T cells to ICAM-1 under two different functional contexts: alpha(4) integrin signaling during emigration from blood into tissues and CD3 signaling during adhesion to APCs and target cells. Cross-linking either alpha(4) integrin or CD3 on Jurkat T cells induced adhesion to ICAM-1 of comparable strength. Adhesion was dependent on phosphatidylinositol (PI) 3-kinase but not p44/42 mitogen-activated protein kinase (extracellular regulated kinase 1/2), because it was inhibited by wortmannin and LY294002 but not U0126. These data suggest that PI 3-kinase is a ubiquitous regulator of beta(2) integrin-mediated adhesion. A distinct morphological change consisting of Jurkat cell spreading and extension of filopodia was induced by alpha(4) integrin signaling. In contrast, CD3 induced radial rings of cortical actin polymerization. Inhibitors of PI 3-kinase and extracellular regulated kinase 1/2 did not affect alpha(4) integrin-induced rearrangement of the actin cytoskeleton, but treatment with ionomycin, a Ca(2+) ionophore, modulated cell morphology by reducing filopodia and promoting lamellipodia formation. Qualitatively similar morphological and adhesive changes to those observed with Jurkat cells were observed following alpha(4) integrin or CD3 stimulation of human peripheral blood T cells.     

Integrin activation involves a conformational change in the alpha 1 helix of the beta subunit A-domain

The ligand-binding region of integrin beta subunits contains a von Willebrand factor type A-domain: an alpha/beta "Rossmann" fold containing a metal ion-dependent adhesion site (MIDAS) on its top face. Although there is evidence to suggest that the betaA-domain undergoes changes in tertiary structure during receptor activation, the identity of the secondary structure elements that change position is unknown. The mAb 12G10 recognizes a unique cation-regulated epitope on the beta(1) A-domain, induction of which parallels the activation state of the integrin (i.e. competency for ligand recognition). The ability of Mn(2+) and Mg(2+) to stimulate 12G10 binding is abrogated by mutation of the MIDAS motif, demonstrating that the MIDAS is a Mn(2+)/Mg(2+) binding site and that occupancy of this site induces conformational changes in the A-domain. The cation-regulated region of the 12G10 epitope maps to Arg(154)/Arg(155) in the alpha1 helix. Our results demonstrate that the alpha1 helix undergoes conformational alterations during integrin activation and suggest that Mn(2+) acts as a potent activator of beta(1) integrins because it can promote a shift in the position of this helix. The mechanism of beta subunit A-domain activation appears to be distinct from that of the A-domains found in some integrin alpha subunits.     

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.     

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.     

Kinetic and mechanical basis of rolling through an integrin and novel Ca2+-dependent rolling and Mg2+-dependent firm adhesion modalities for the alpha 4 beta 7-MAdCAM-1 interaction.

We studied interactions in shear flow of cells bearing integrins alpha4beta1 or alpha4beta7 with VCAM-1 and MAdCAM-1 substrates in different divalent cations. Interestingly, Ca(2+) was essential for tethering in flow and rolling interactions through both alpha4 integrins. Mg(2+) promoted firm adhesion of alpha4beta7-expressing cells on MAdCAM-1 but with much lower tethering efficiency in shear flow. The k(off) degrees of 1.28 s(-1) and resistance of the receptor-ligand bond to force (estimated as a bond interaction distance or sigma) for transient tethers on MAdCAM-1 were similar to values for E- and P-selectins. By contrast to results in Ca(2+) or Ca(2+) + Mg(2+), in Mg(2+) the alpha4beta7-MAdCAM-1 k(off) degrees decreased 20-fold to 0.046 s(-1), and the bond was weaker, providing an explanation for the finding of firm adhesion under these conditions. Shear enhanced tethering to MAdCAM-1, thereby contributing to the stability of rolling. Comparisons to selectins demonstrate that the kinetic and mechanical properties of the alpha4beta7 integrin are well suited to its intermediate position in adhesion cascades, in which it bridges rapid rolling through selectins to firm adhesion through beta2 integrins.     

A minimized human integrin alpha(5)beta(1) that retains ligand recognition

Two isolated recombinant fragments from human integrin alpha(5)beta(1) encompassing the FG-GAP repeats III to VII of alpha(5) and the insertion-type domain from beta(1), respectively, are structurally well defined in solution, based on CD evidence. Divalent cation binding induces a conformational adaptation that is achieved by Ca(2+) or Mg(2+) (or Mn(2+)) with alpha(5) and only by Mg(2+) (or Mn(2+)) with beta(1). Mn(2+) bound to beta(1) is highly hydrated ( approximately 3 water molecules), based on water NMR relaxation, in agreement with a metal ion-dependent adhesion site-type metal coordination. Each fragment saturated with Mg(2+) (or Mn(2+)) binds a recombinant fibronectin ligand in an RGD-dependent manner. A conformational rearrangement is induced on the fibronectin ligand upon binding to the alpha(5), but not to the beta(1) fragment, based on CD. Ligand binding results in metal ion displacement from beta(1). Both alpha(5) and beta(1) fragments form a stable heterodimer (alpha(5)beta(1) mini-integrin) that retains ligand recognition to form a 1:1:1 ternary complex, in the presence of Mg(2+), and induces a specific conformational adaptation of the fibronectin ligand. A two-site model for RGD binding to both alpha and beta integrin components is inferred from our data using low molecular weight RGD mimetics.     

The alpha(4) integrin subunit Tyr(187) has a key role in alpha(4)beta(7)-dependent cell adhesion

The integrin alpha(4)beta(7) is the cell adhesion receptor for the mucosal vascular addressin MAdCAM-1, and this interaction is dominant in lymphocyte homing to Peyer's patch high endothelial venules, and plays key roles in lymphocyte recruitment at sites of inflammation. To identify alpha(4) subunit amino acids important for alpha(4)beta(7)/MAdCAM-1 interaction, we expressed mutant alpha(4) and wild type beta(7) chains in K562 cells and analyzed the effect of the mutations on cell adhesion to a soluble MAdCAM-1 (sMAdCAM-1-Ig). Transfectants expressing mutated alpha(4) at Tyr(187) displayed a substantial decrease in adhesion to this ligand, which was associated with a reduced alpha(4)beta(7)/sMAdCAM-1-Ig interaction, as determined by soluble binding assays. Addition of Mn(2+) to the adhesion assays did not restore the impaired adhesion. Mutations at alpha(4) Gln(152)Asp(153) also affected transfectant adhesion to sMAdCAM-1-Ig, but did not involve an alteration of alpha(4)beta(7)/MAdCAM-1 binding, and adhesion was restored by Mn(2+). Instead, mutations at alpha(4) Asn(123)Glu(124) did not affect this adhesion. Mutation of alpha(4) Tyr(187) abolished alpha(4)beta(7)-mediated cell adhesion to CS-1/fibronectin, an additional ligand for alpha(4)beta(7), while alpha(4) Gln(152)Asp(153) transfectant mutants showed a reduced adhesion. These results identify alpha(4) Tyr(187) as a key residue during receptor alpha(4)beta(7)/ligand interactions, indicating that it plays important roles in alpha(4)beta(7)-mediated leukocyte adhesion, and provide a potential target for therapeutic intervention in several inflammatory pathologies.     

The relative influence of metal ion binding sites in the I-like domain and the interface with the hybrid domain on rolling and firm adhesion by integrin alpha4beta7

We examined the effect of conformational change at the beta(7) I-like/hybrid domain interface on regulating the transition between rolling and firm adhesion by integrin alpha(4)beta(7). An N-glycosylation site was introduced into the I-like/hybrid domain interface to act as a wedge and to stabilize the open conformation of this interface and hence the open conformation of the alpha(4) beta(7) headpiece. Wild-type alpha(4)beta(7) mediates rolling adhesion in Ca(2+) and Ca(2+)/Mg(2+) but firm adhesion in Mg(2+) and Mn(2+). Stabilizing the open headpiece resulted in firm adhesion in all divalent cations. The interaction between metal binding sites in the I-like domain and the interface with the hybrid domain was examined in double mutants. Changes at these two sites can either counterbalance one another or be additive, emphasizing mutuality and the importance of multiple interfaces in integrin regulation. A double mutant with counterbalancing deactivating ligand-induced metal ion binding site (LIMBS) and activating wedge mutations could still be activated by Mn(2+), confirming the importance of the adjacent to metal ion-dependent adhesion site (ADMIDAS) in integrin activation by Mn(2+). Overall, the results demonstrate the importance of headpiece allostery in the conversion of rolling to firm adhesion.     

Hierarchy of ADAM12 binding to integrins in tumor cells

ADAMs (a disintegrin and metalloprotease) comprise a family of cell surface proteins with protease and cell-binding activities. Using different forms and fragments of ADAM12 as substrates in cell adhesion and spreading assays, we demonstrated that alpha9beta1 integrin is the main receptor for ADAM12. However, when alpha9beta1 integrin is not expressed--as in many carcinoma cells--other members of the beta1 integrin family can replace its ligand binding activity. In attachment assays, the recombinant disintegrin domain of ADAM12 only supported alpha9 integrin-dependent tumor cell attachment, whereas full-length ADAM12 supported attachment via alpha9 integrin and other integrin receptors. Cells that attached to full-length ADAM12 in an alpha9 integrin-dependent manner also attached to ADAM12 in which the putative alpha9beta1 integrin-binding motif in the disintegrin domain had been mutated. This attachment was mediated through use of an alternate beta1 integrin. We also found that cell spreading in response to ADAM12 is dependent on the apparent level of integrin activation. Binding of cells to ADAM12 via the alpha9beta1 integrin was Mn(2+)-independent and resulted in attachment of cells with a rounded morphology; attachment of cells with a spread morphology required further activation of the alpha9beta1 integrin. We demonstrated that phosphoinositide-3-kinase appears to be central in regulating alpha9beta1 integrin cell spreading activity in response to ADAM12.     

A cell-free electrochemiluminescence assay for measuring beta1-integrin-ligand interactions

We have developed a cell-free assay for binding of solubilized beta1 integrins to their physiologically relevant ligands using an electrochemiluminescent detection method. The method utilizes ruthenium-conjugated monoclonal antibodies for detection of either purified integrins or, more conveniently, integrin-expressing cell lysates, which are captured on beads coated with extracellular matrix or vascular ligand proteins. For the interaction of alpha1beta1 integrin with collagen IV, a signal of 10-fold over background was generated with samples containing only 10 ng (0.05 pmol) of integrin. This interaction is cation-dependent and can be inhibited by blocking antibodies to the alpha1 subunit. The method was extended to studies of ligand binding by integrins alpha2beta1, alpha4beta1, alpha5beta1, and alpha6beta1. For each integrin-ligand pair, the specificity of the interaction was verified with neutralizing antibodies against the specific integrin. The specific binding signal correlated with the activating ability of the labeled antibody used for detection, although the ability of divalent cations (Mn2+, Mg2+, Ca2+) to support integrin-ligand binding varied dramatically among the various integrin-ligand pairs. The assay provides a simple method for investigating integrin-ligand interactions without avidity and/or signaling effects which can complicate conventional cell-based assay methods.     

Integrin alphavbeta6 mediates HT29-D4 cell adhesion to MMP-processed fibrinogen in the presence of Mn2+

Mn(2+) was found to induce adhesion of HT29-D4 adenoma carcinoma cells to fibrinogen (Fb). This was independent of the expression of the beta3 integrin subunit and involved endogenous alphavbeta6 but not alphavbeta5 integrin. Thus, addition of Mn(2+) led to a change in integrin alphavbeta6 specificity. Furthermore, Mn(2+) was found to strongly activate the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in the HT29-D4 cell line. As a MAPK inhibitor strongly reduced the Mn(2+)-induced cell adhesion to Fb, it is suggested that a link between MAPK activation and cell adhesion to Fb exists. Both expression and activity of matrix metalloproteinase-9 (MMP-9) were enhanced by Mn(2+) and this led to Fb processing. MMP inhibitors prevented Mn(2+)-mediated cell adhesion to Fb, leading us to suggest that Mn(2+) promoted convergent changes in integrin alphavbeta6 conformation and Fb structure through activation of ERK/MAPK and MMP-9. Finally, we found that Mn(2+) and activators of the ERK pathway cooperated in HT29-D4 cell adhesion to Fb. Such a process may be involved in bone metastasis of some cancer cells.     

A small molecule agonist of an integrin, alphaLbeta2

The binding of integrin alpha(L)beta(2) to its ligand intercellular adhesion molecule-1 is required for immune responses and leukocyte trafficking. Small molecule antagonists of alpha(L)beta(2) are under intense investigation as potential anti-inflammatory drugs. We describe for the first time a small molecule integrin agonist. A previously described alpha/beta I allosteric inhibitor, compound 4, functions as an agonist of alpha(L)beta(2) in Ca(2+) and Mg(2+)and as an antagonist in Mn(2+). We have characterized the mechanism of activation and its competitive and noncompetitive inhibition by different compounds. Although it stimulates ligand binding, compound 4 nonetheless inhibits lymphocyte transendothelial migration. Agonism by compound 4 results in accumulation of alpha(L)beta(2) in the uropod, extreme uropod elongation, and defective de-adhesion. Small molecule integrin agonists open up novel therapeutic possibilities.     

Effect of divalent cations on the affinity and selectivity of alpha4 integrins towards the integrin ligands vascular cell adhesion molecule-1 and mucosal addressin cell adhesion molecule-1: Ca2+ activation of integrin alpha4beta1 confers a distinct ligand specificity

A microtiter plate assay measuring the binding of cells expressing integrins alpha4beta1 or alpha4beta7 to VCAM-1 and MAdCAM-1, expressed as Ig fusion proteins, was used to explore the interplay between the variables of integrin beta-chain, identity and density of ligand, and identity and concentration of activating cations. Both Mn2+ and Mg2+ supported binding of either integrin to either ligand. Ca2+ supported only the binding of alpha4beta1 to VCAM-Ig. Cation concentrations required for half-maximal binding (EC50) ranged from 0.8-280 microM for Mn2+ and 0.8-30 mM for Mg2+, being thus 2-3 logs lower for Mn2+ compared to Mg2+ independent of ligand. EC50 values for binding of alpha4beta1 to VCAM-Ig were 30-45-fold lower compared to MAdCAM-Ig, while alpha4beta7 showed an opposite 3-15-fold selectivity for MAdCAM-Ig over VCAM-Ig. The density of ligand required for adhesion via alpha4beta1 was markedly lower with Mn2+ versus Mg2+, and with VCAM-Ig versus MAdCAM-Ig. These results were interpreted in terms of a coupled equilibrium model, in which binding of activating metal ions and of integrin ligands each stabilizes activated integrin. We conclude that Mn2+ and Mg2+ bind to common regulatory sites with different affinities, producing similar activated states of the integrin. The resulting activated alpha4beta1 binds more strongly to VCAM-Ig versus MAdCAM-Ig by 30-45-fold, while similarly activated alpha4beta7 binds more strongly to MAdCAM-Ig versus VCAM-Ig by 3-15-fold. Inhibition studies showed that Ca2+ also binds to regulatory sites on both integrins. However, the Ca2+-activated state of alpha4beta1 is distinct from that achieved by Mn2+ and Mg2+, possessing increased selectivity for binding to VCAM-1 versus MAdCAM-1.     

Interactions of thrombospondins with alpha4beta1 integrin and CD47 differentially modulate T cell behavior

Thrombospondin (TSP)-1 has been reported to modulate T cell behavior both positively and negatively. We found that these opposing responses arise from interactions of TSP1 with two different T cell receptors. The integrin alpha4beta1 recognizes an LDVP sequence in the NH2-terminal domain of TSP1 and was required for stimulation of T cell adhesion, chemotaxis, and matrix metalloproteinase gene expression by TSP1. Recognition of TSP1 by T cells depended on the activation state of alpha4beta1 integrin, and TSP1 inhibited interaction of activated alpha4beta1 integrin on T cells with its counter receptor vascular cell adhesion molecule-1. The alpha4beta1 integrin recognition site is conserved in TSP2. A recombinant piece of TSP2 containing this sequence replicated the alpha4beta1 integrin-dependent activities of TSP1. The beta1 integrin recognition sites in TSP1, however, were neither necessary nor sufficient for inhibition of T cell proliferation and T cell antigen receptor signaling by TSP1. A second TSP1 receptor, CD47, was not required for some stimulatory responses to TSP1 but played a significant role in its T cell antigen receptor antagonist and antiproliferative activities. Modulating the relative expression or function of these two TSP receptors could therefore alter the direction or magnitude of T cell responses to TSPs.     

Vascular endothelial-junctional adhesion molecule (VE-JAM)/JAM 2 interacts with T, NK, and dendritic cells through JAM 3

Screening expressed sequence tag databases for endothelial-specific homologs to human junctional adhesion molecule (JAM) and A33-Ag, we identified a protein of 298 aa that represents the recently described vascular endothelial-JAM (VE-JAM)/JAM 2. We confirmed VE-JAM/JAM 2 expression to be restricted to the high endothelial venule of tonsil and lymph nodes, and we further expanded the localization to the endothelium of arterioles in and around inflammatory and tumor foci. In our functional characterizations of VE-JAM/JAM 2, we discovered that it can function as an adhesive ligand for the T cell line J45 and can interact with GM-CSF/IL-4-derived peripheral blood dendritic cells, circulating CD56(+) NK cells, circulating CD56(+)CD3(+) NK/T cells, and circulating CD56(+)CD3(+)CD8(+) cytolytic T cells. In the course of our studies, we also isolated and characterized the functional VE-JAM/JAM 2 receptor, which, upon cloning, turned out to be a submitted sequence representing JAM 3 (accession number NP 113658). With these understandings, we have characterized a protein-interacting pair that can be important in the role of T, NK, and dendritic cell trafficking and inflammation.     

The integrin alpha 6 beta 4 is a laminin receptor

In this study, the putative laminin receptor function of the alpha 6 beta 4 integrin was assessed. For this purpose, we used a human cell line, referred to as clone A, that was derived from a highly invasive, colon adenocarcinoma. This cell line, which expresses the alpha 6 beta 4 integrin, adheres to the E8 and not to the P1 fragment of laminin. The adhesion of clone A cells to laminin is extremely rapid with half-maximal adhesion observed at 5 min after plating. Adhesion to laminin is blocked by GoH3, and alpha 6 specific antibody (60% inhibition), as well as by A9, a beta 4 specific antibody (30% inhibition). Most importantly, we demonstrate that alpha 6 beta 4 binds specifically to laminin-Sepharose columns in the presence of either Mg2+ or Mn2+ and it is eluted from these columns with EDTA but not with NaCl. The alpha 6 beta 4 integrin does not bind to collagen-Sepharose, but the alpha 2 beta 1 integrin does bind. Clone A cells do not express alpha 6 beta 1 as evidenced by the following observations: (a) no beta 1 integrin is detected in beta 1 immunoblots of GoH3 immunoprecipitates; and (b) no alpha 6 beta 1 integrin is seen in GoH3 immunoprecipitates of clone A extracts that had been immunodepleted of all beta 4 containing integrin using the A9 antibody. These data establish that laminin is a ligand for the alpha 6 beta 4 integrin and that this integrin can function as a laminin receptor independently of alpha 6 beta 1.     

The small GTPase Rap1 is required for Mn(2+)- and antibody-induced LFA-1- and VLA-4-mediated cell adhesion

In T-lymphocytes the Ras-like small GTPase Rap1 plays an essential role in stimulus-induced inside-out activation of integrin LFA-1 (alpha(L)beta(2)) and VLA-4 (alpha(4)beta(1)). Here we show that Rap1 is also involved in the direct activation of these integrins by divalent cations or activating antibodies. Inhibition of Rap1 either by Rap GTPase-activating protein (RapGAP) or the Rap1 binding domain of RalGDS abolished both Mn(2+)- and KIM185 (anti-LFA-1)-induced LFA-1-mediated cell adhesion to intercellular adhesion molecule 1. Mn(2+)- and TS2/16 (anti-VLA-4)-induced VLA-4-mediated adhesion were inhibited as well. Interestingly, both Mn(2+), KIM185 and TS2/16 failed to induce elevated levels of Rap1GTP. These findings indicate that available levels of GTP-bound Rap1 are required for the direct activation of LFA-1 and VLA-4. Pharmacological inhibition studies demonstrated that both Mn(2+)- and KIM185-induced adhesion as well as Rap1-induced adhesion require intracellular calcium but not signaling activity of the MEK-ERK pathway. Moreover, functional calmodulin signaling was shown to be a prerequisite for Rap1-induced adhesion. From these results we conclude that in addition to stimulus-induced inside-out activation of integrins, active Rap1 is required for cell adhesion induced by direct activation of integrins LFA-1 and VLA-4. We suggest that Rap1 determines the functional availability of integrins for productive binding to integrin ligands.     

Evidence that ligand and metal ion binding to integrin alpha 4beta 1 are regulated through a coupled equilibrium

We have used the highly selective alpha(4)beta(1) inhibitor 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino)-pentanoylamino]-butyric acid (BIO7662) as a model ligand to study alpha(4)beta(1) integrin-ligand interactions on Jurkat cells. Binding of [(35)S]BIO7662 to Jurkat cells was dependent on the presence of divalent cations and could be blocked by treatment with an excess of unlabeled inhibitor or with EDTA. K(D) values for the binding of BIO7662 to Mn(2+)-activated alpha(4)beta(1) and to the nonactivated state of the integrin that exists in 1 mm Mg(2+), 1 mm Ca(2+) were <10 pm, indicating that it has a high affinity for both activated and nonactivated integrin. No binding was observed on alpha(4)beta(1) negative cells. Through an analysis of the metal ion dependences of ligand binding, several unexpected findings about alpha(4)beta(1) function were made. First, we observed that Ca(2+) binding to alpha(4)beta(1) was stimulated by the addition of BIO7662. From solution binding studies on purified alpha(4)beta(1), two types of Ca(2+)-binding sites were identified, one dependent upon and the other independent of BIO7662 binding. Second, we observed that the metal ion dependence of ligand binding was affected by the affinity of the ligand for alpha(4)beta(1). ED(50) values for the metal ion dependence of the binding of BIO7762 and the binding of a lower affinity ligand, BIO1211, differed by 2-fold for Mn(2+), 30-fold for Mg(2+), and >1000-fold for Ca(2+). Low Ca(2+) (ED(50) = 5-10 microm) stimulated the binding of BIO7662 to alpha(4)beta(1). The effects of microm Ca(2+) closely resembled the effects of Mn(2+) on alpha(4)beta(1) function. Third, we observed that the rate of BIO7662 binding was dependent on the metal ion concentration and that the ED(50) for the metal ion dependence of BIO7662 binding was affected by the concentration of the BIO7662. These studies point to an even more complex interplay between metal ion and ligand binding than previously appreciated and provide evidence for a three-component coupled equilibrium model for metal ion-dependent binding of ligands to alpha(4)beta(1).     

Nectin-like molecule-5/Tage4 enhances cell migration in an integrin-dependent, Nectin-3-independent manner

Cell migration plays roles in invasion of transformed cells and scattering of embryonic mesenchymal cells into surrounding tissues. We have found that Ig-like Necl-5/Tage4 is up-regulated in NIH3T3 cells transformed by an oncogenic Ras (V12Ras-NIH3T3 cells) and heterophilically trans-interacts with a Ca(2+)-independent Ig-like cell adhesion molecule nectin-3, eventually enhancing their intercellular motility. We show here that Necl-5 furthermore enhances cell migration in a nectin-3-independent manner. Studies using L fibroblasts expressing various mutants of Necl-5, NIH3T3 cells, and V12Ras-NIH3T3 cells have revealed that Necl-5 enhances serum- and platelet-derived growth factor-induced cell migration. The extracellular region of Necl-5 is necessary for directional cell migration, but not for random cell motility. The cytoplasmic region of Necl-5 is necessary for both directional and random cell movement. Necl-5 colocalizes with integrin alpha(V)beta(3) at leading edges of migrating cells. Analyses using an inhibitor or an activator of integrin alpha(V)beta(3) or a dominant negative mutant of Necl-5 have shown the functional association of Necl-5 with integrin alpha(V)beta(3) in cell motility. Cdc42 and Rac small G proteins are activated by the action of Necl-5 and required for the serum-induced, Necl-5-enhanced cell motility. These results indicate that Necl-5 regulates serum- and platelet-derived growth factor-induced cell migration in an integrin-dependent, nectin-3-independent manner, when cells do not contact other cells. We furthermore show here that enhanced motility and metastasis of V12Ras-NIH3T3 cells are at least partly the result of up-regulated Necl-5.