Human B lymphocytes define an alternative mechanism of adhesion to fibronectin. The interaction of the alpha 4 beta 1 integrin with the LHGPEILDVPST sequence of the type III connecting segment is sufficient to promote cell attachment

In this report we have studied the mechanism of human B lymphocyte adhesion to fibronectin and to proteolytic fragments of this protein. B cells adhered to fibronectin and to a 38-kDa fragment, derived from the A chain, containing the Hep II domain and most of the type III connecting segment, IIICS, of fibronectin. Cells did not bind to an 80-kDa fragment containing the RGD adhesive sequence of fibronectin. Attachment to fibronectin or to the 38-kDa fragment was not affected by the 80-kDa fragment, the GRGDSPC synthetic peptide, or by a mAb specific for the alpha chain of the RGD-dependent fibronectin receptor, alpha 5 beta 1. However, B cell adhesion to fibronectin was inhibited by the synthetic peptides CS-1, comprising the first 25 amino acids of IIICS and B12, containing the sequence LHGPEILDVPST of CS-1 (residues 14-25). Moreover, this sequence was shown to be sufficient to induce stable cell adhesion when coated on plastic surfaces. A mAb specific for the alpha-subunit of the alpha 4 beta 1 integrin, completely inhibited B cell attachment to B12, CS-1, 38 kDa, and fibronectin coated substrata. These data clearly indicate that adhesion of B lymphocytes to fibronectin is exclusively mediated by the interaction of alpha 4 beta 1 with residues 14-25 of the IIICS region in fibronectin. Therefore this interaction constitutes an alternative pathway of adhesion to fibronectin, independent of RGD and alpha 5 beta 1.     

The α4β1 Fibronectin Ligands CS-19 HEP II,and RGD Induce Different Intracellular Events in B Lymphoid Cells. Comparison with the Effects of the Endothelial Ligand V CAM-1

The lymphocyte integrin α4β1 is the receptor for the Hep II domain and CS-1 site in fibronectin (Fn) as well as for VCAM-1. We recently showed that upon activation with anti-β1 mAb TS2/16, α4β1 also recognizes the RGD Fn sequence. To determine the physiological role of these multiple interactions, we have now studied some intracellular events induced by “resting” and activated α4β1 binding to its different ligands. Analyses of actin and tubulin reorganization upon adhesion of B lymphoid cells to Fn fragments or VCAM-1 showed that VCAM-1, a 38 kDa fragment (Hep II+CS-1), and the CS-1 synthetic peptide induced formation of transient cytoplasmic projections; however, cells attached to a 58 kDa (Hep II) or 80 kDa (RGD) fragments remained rounded. Using transfilter assays, we showed that VCAM-1, 38 kDa and CS-1 also induced dose-dependent B cell migration mediated by α4β1. Furthermore, these three ligands, but not the 80 kDa fragment or a synthetic peptide (H1) containing a sequence from Hep II shown to bind α4β1, induced tyrosine phosphorylation of a 110 kDa protein. Activation of α4β1 with TS2/16 inhibited the cytoplasmic protrusions and cell migration but did not affect the pattern of phosphorylation. Our results indicate that the various α4β1 ligands induce different cellular responses. Most importantly they show that α4β1 interaction with CS-1 is sufficient to trigger intracellular events in B cells. Furthermore, they suggest a regulation by the activation form of the receptor as well as by the ligand in events involving lymphocyte adhesion and migration.     

Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin

Using mAb technology (Wayner, E. A., W. G. Carter, R. Piotrowicz, and T. J. Kunicki. 1988. J. Cell Biol. 107:1881-1891), we have identified a new fibronectin receptor that is identical to the integrin receptor alpha 4 beta 1. mAbs P3E3, P4C2, and P4G9 recognized epitopes on the alpha 4 subunit and completely inhibited the adhesion of peripheral blood and cultured T lymphocytes to a 38-kD tryptic fragment of plasma fibronectin containing the carboxy-terminal Heparin II domain and part of the type III connecting segment (IIICS). The ligand in IIICS for alpha 4 beta 1 was the CS-1 region previously defined as an adhesion site for melanoma cells. The functionally defined mAbs to alpha 4 partially inhibited T lymphocyte adhesion to intact plasma fibronectin and had no effect on their attachment to an 80-kD tryptic fragment containing the RGD (arg-gly-asp) adhesion sequence. mAbs (P1D6 and P1F8) to the previously described fibronectin receptor, alpha 5 beta 1, completely inhibited T lymphocyte adhesion to the 80-kD fragment but had no effect on their attachment to the 38-kD fragment or to CS-1. Both alpha 4 beta 1 and alpha 5 beta 1 localized to focal adhesions when fibroblasts that express these receptors were grown on fibronectin-coated surfaces. These findings demonstrated a specific interaction of both receptors with fibronectin at focal contacts. In conclusion, these findings show clearly that cultured T lymphocytes use two independent receptors during attachment to fibronectin and that (a) alpha 5 beta 1 is the receptor for the RGD containing cell adhesion domain, and (b) alpha 4 beta 1 is the receptor for a carboxy-terminal cell adhesion region containing the Heparin II and IIICS domains. Furthermore, these data also show that T lymphocytes express a clear preference for a region of molecular heterogeneity in IIICS (CS-1) generated by alternative splicing of fibronectin pre-mRNA and that alpha 4 beta 1 is the receptor for this adhesion site.     

Alpha4beta1 integrin/ligand interaction inhibits alpha5beta1-induced stress fibers and focal adhesions via down-regulation of RhoA and induces melanoma cell migration

We have studied the function of the Hep III fibronectin domain in the cytoskeletal response initiated by alpha5beta1 integrin-mediated adhesion. Melanoma cells formed stress fibers and focal adhesions on the RGD-containing FNIII7-10 fragment. Coimmobilization of FNIII4-5, a fragment spanning Hep III and containing the alpha4beta1 ligand H2 with FNIII7-10, or addition of soluble FNIII4-5 to cells preattached to FNIII7-10, inhibited stress fibers and induced cytoplasmic protrusions. This effect involved alpha4beta1 since: 1) mutations in H2 reverted the inhibition; 2) other alpha4beta1 ligands (CS-1, VCAM-1), an anti-alpha4 mAb, or alpha4 expression in HeLa cells inhibited stress fibers. This activity was apparently cryptic in fibronectin or large fibronectin fragments, but exposed upon proteolytic degradation. Indeed purified peptic fragments containing H2, inhibited stress fibers when mixed with FNIII7-10 or fibronectin. RhoA activation with LPA or transfection with V14RhoA reverted the inhibitory effect and induced stress fibers on FNIII7-10+FNIII4-5. Furthermore, addition of alpha4beta1 ligands to FNIII7-10, down-regulated RhoA and activated p190RhoGAP, which localized to cytoplasmic protrusions. alpha4beta1/ligand interaction induced cell migration, monitored by video microscopy and wound healing assays. These data indicate that alpha4beta1 provides an antagonistic signal to alpha5beta1 by interfering with the RhoA activation pathway and this leads to melanoma cell migration.     

Adhesion of osteosarcoma cells to the 70-kDa core region of thrombospondin-1 is mediated by the alpha 4 beta 1 integrin

Thrombospondin-1 (TSP-1) is an extracellular glycoprotein that is involved in a variety of physiological processes such as tumor cell adhesion, invasion, and metastasis. It has been hypothesized that TSP-1 provides an adhesive matrix for osteosarcoma cells. Here we present data showing that TSP-1 can promote cell substrate adhesion to U2OS and SAOS cells through the alpha 4 beta 1 integrin. The dose-dependent adhesion to TSP-1 was inhibited by anti-integrin antibodies directed against the alpha 4 or beta 1 subunit, but not by control antibodies against other integrins. To localize the potential alpha 4 beta 1-binding site within the TSP-1 molecule, the protein was subjected to limited proteolysis with chymotrypsin in the absence of calcium. The stable 70-kDa core fragment produced under these conditions promoted alpha 4 beta 1-dependent osteosarcoma cell adhesion in a manner similar to that of the intact protein. Moreover adhesion experiments with neutralizing antibodies revealed that the adhesion was totally dependent on the alpha 4 beta 1 interaction. Further blocking experiments with potential inhibitory peptides revealed that the alpha 4 beta 1-mediated adhesion was not influenced by peptides containing the RGD sequence. Attachment to the 70-kDa fragment was strongly inhibited by the CS-1 peptide, which represents the most active recognition domain for alpha 4 beta 1 integrin in fibronectin. The present data provide evidence that TSP-1 contains an alpha 4 beta 1 integrin-binding site within the 70-kDa core region.     

The integrin alpha(9)beta(1) binds to a novel recognition sequence (SVVYGLR) in the thrombin-cleaved amino-terminal fragment of osteopontin

The integrin alpha(9)beta(1) mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH(2)-terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for alpha(9)beta(1). In this report, we used site-directed mutagenesis and synthetic peptides to identify the alpha(9)beta(1) recognition sequence in osteopontin. alpha(9)-transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH(2)-terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-alpha(9) monoclonal antibody Y9A2, indicating the presence of a non-RGD alpha(9)beta(1) recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on alpha(9)beta(1)-mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr(165), blocked alpha(9)beta(1)-mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin beta(1) subunit on alpha(9)-transfected, but not on mock-transfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to alpha(9)beta(1) and is responsible for alpha(9)beta(1)-mediated cell adhesion to the NH(2)-terminal fragment of osteopontin.     

Molecular basis of ligand recognition by integrin alpha5beta 1. II. Specificity of arg-gly-Asp binding is determined by Trp157 OF THE alpha subunit

Different beta(1) integrins bind Arg-Gly-Asp (RGD) peptides with differing specificities, suggesting a role for residues in the alpha subunit in determining ligand specificity. Integrin alpha(5)beta(1) has been shown to bind with high affinity to peptides containing an Arg-Gly-Asp-Gly-Trp (RGDGW) sequence but with relatively low affinity to other RGD peptides. The residues within the ligand-binding pocket that determine this specificity are currently unknown. A cyclic peptide containing the RGDGW sequence was found to strongly perturb the binding of the anti-alpha(5) monoclonal antibody (mAb) 16 to alpha(5)beta(1). In contrast, RGD peptides lacking the tryptophan residue acted as weak inhibitors of mAb 16 binding. The epitope of mAb 16 has previously been localized to a region of the alpha(5) subunit that contains Ser(156)-Trp(157). Mutation of Trp(157) (but not of Ser(156) or surrounding residues) to alanine blocked recognition of mAb 16 and perturbed the high affinity binding of RGDGW-containing peptides to alpha(5)beta(1). The same mutation also abrogated recognition of the alpha(5)beta(1)-specific ligand peptide Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Based on these findings, we propose that Trp(157) of alpha(5) participates in a hydrophobic interaction with the tryptophan residue in RGDGW, and that this interaction determines the specificity of alpha(5)beta(1) for RGDGW-containing peptides. Since the RGD sequence is recognized predominantly by amino acid residues on the beta(1) subunit, our results suggest that Trp(157) of alpha(5) must lie very close to these residues. Our findings therefore provide new insights into the structure of the ligand-binding pocket of alpha(5)beta(1).     

Coordinate role for cell surface chondroitin sulfate proteoglycan and alpha 4 beta 1 integrin in mediating melanoma cell adhesion to fibronectin

Cellular recognition and adhesion to the extracellular matrix (ECM) has a complex molecular basis, involving both integrins and cell surface proteoglycans (PG). The current studies have used specific inhibitors of chondroitin sulfate proteoglycan (CSPG) synthesis along with anti-alpha 4 integrin subunit monoclonal antibodies to demonstrate that human melanoma cell adhesion to an A-chain derived, 33-kD carboxyl-terminal heparin binding fragment of human plasma fibronectin (FN) involves both cell surface CSPG and alpha 4 beta 1 integrin. A direct role for cell surface CSPG in mediating melanoma cell adhesion to this FN fragment was demonstrated by the identification of a cationic synthetic peptide, termed FN-C/H-III, within the fragment. FN-C/H-III is located close to the amino terminal end of the fragment, representing residues #1721-1736 of intact FN. FN-C/H-III binds CSPG directly, can inhibit CSPG binding to the fragment, and promotes melanoma cell adhesion by a CSPG-dependent, alpha 4 beta 1 integrin-independent mechanism. A scrambled version of FN-C/H-III does not inhibit CSPG binding or cell adhesion to the fragment or to FN-C/H-III, indicating that the primary sequence of FN-C/H-III is important for its biological properties. Previous studies have identified three other synthetic peptides from within this 33-kD FN fragment that promote cell adhesion by an arginyl-glycyl-aspartic acid (RGD) independent mechanism. Two of these synthetic peptides (FN-C/H-I and FN-C/H-II) bind heparin and promote cell adhesion, implicating cell surface PG in mediating cellular recognition of these two peptides. Additionally, a third synthetic peptide, CS1, is located in close proximity to FN-C/H-I and FN-C/H-II and it promotes cell adhesion by an alpha 4 beta 1 integrin-dependent mechanism. In contrast to FN-C/H-III, cellular recognition of these three peptides involved contributions from both CSPG and alpha 4 integrin subunits. Of particular importance are observations demonstrating that CS1-mediated melanoma cell adhesion could be inhibited by interfering with CSPG synthesis or expression. Since CS1 does not bind CSPG, the results suggest that CSPG may modify the function and/or activity of alpha 4 beta 1 integrin on the surface of human melanoma cells. Together, these results support a model in which the PG and integrin binding sites within the 33-kD fragment may act in concert to focus these two cell adhesion receptors into close proximity on the cell surface, thereby influencing initial cellular recognition events that contribute to melanoma cell adhesion on this fragment.     

Recognition of the N-terminal modules of thrombospondin-1 and thrombospondin-2 by alpha6beta1 integrin

In addition to its recognition by alpha3beta1 and alpha4beta1 integrins, the N-terminal pentraxin module of thrombospondin-1 is a ligand for alpha6beta1 integrin. alpha6beta1 integrin mediates adhesion of human microvascular endothelial and HT-1080 fibrosarcoma cells to immobilized thrombospondin-1 and recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. alpha6beta1 also mediates chemotaxis of microvascular cells to thrombospondin-1 and thrombospondin-2. Using synthetic peptides, LALERKDHSG was identified as an alpha6beta1-binding sequence in thrombospondin-1. This peptide inhibited alpha6beta1-dependent cell adhesion to thrombospondin-1, thrombospondin-2, and the E8 fragment of murine laminin-1. The Glu residue in this peptide was required for activity, and the corresponding residue (Glu90) in the N-terminal module of thrombospondin-1 was required for its recognition by alpha6beta1, but not by alpha4beta1. alpha6beta1 was also expressed in human umbilical vein endothelial cells; but in these cells, only certain agonists could activate the integrin to recognize thrombospondins. Selective activation of alpha6beta1 integrin in microvascular endothelial cells by the anti-beta1 antibody TS2/16 therefore accounts for their adhesion responses to thrombospondins and explains the distinct functions of alpha4beta1 and alpha6beta1 integrins as thrombospondin receptors in microvascular and large vessel endothelial cells.     

Analysis of the alpha4beta1 integrin-osteopontin interaction

The integrin alpha4beta1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via alpha4beta1 using GST fusion proteins. We show that alpha4beta1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the alpha4beta1 binding region in OPN lies between amino acid residues 125 and 168 (aa125-168). This region contains the central RGD motif of OPN, which also interacts with integrins alphavbeta3, alphavbeta5, alphavbeta1, alpha8beta1, and alpha5beta1. Mutating the RGD motif to RAD had no effect on the interaction with alpha4beta1. To define the binding site the region incorporating aa125-168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via alpha4beta1, aa132-146, and aa153-168; of these only a synthetic peptide, SVVYGLR (aa162-168), derived from aa153-168 was able to inhibit alpha4beta1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of alpha4beta1, and the primary alpha4beta1 binding site within OPN.     

The lymphocyte metalloprotease MDC-L (ADAM 28) is a ligand for the integrin alpha4beta1.

The interaction of lymphocytes with other cells is critical for normal immune surveillance and response. MDC-L (ADAM 28), a member of the ADAM (a disintegrin and metalloprotease) protein family, is expressed on the surface of human lymphocytes. ADAMs possess a disintegrin-like domain similar in sequence to small non-enzymatic snake venom peptides that act as integrin antagonists. We report here that the disintegrin domain of MDC-L is recognized by the leukocyte integrin alpha(4)beta(1). Recombinant Fc fusion proteins possessing the disintegrin domain of MDC-L supported adhesion of the T-lymphoma cell line, Jurkat, in a concentration- and divalent cation-dependent manner. Adhesion of Jurkat cells to the disintegrin domain of MDC-L was inhibited by an anti-MDC-L monoclonal antibody (mAb), Dis1-1. The epitope for mAb Dis1-1 was localized within 59 residues of the disintegrin domain. Recombinant expression of this 59-residue fragment of the disintegrin domain also supported cell adhesion. Adhesion of Jurkat cells to the MDC-L disintegrin domain was specifically inhibited by anti-alpha(4) and anti-beta(1) function-blocking mAbs. Furthermore, adhesion of various cell lines to MDC-L correlated with expression of the integrin alpha(4)-subunit. Transfected K562 cells expressing alpha(4)beta(1) adhered to the disintegrin domain in contrast to non-transfected K562 cells. We further investigated the binding of recombinant MDC-L disintegrin domain (rDis-Fc) in solution. The rDis-Fc was found to bind to Jurkat cells in solution in a concentration-dependent and saturable manner. Both adhesion and solution binding of rDis-Fc was inhibited by the alpha(4)beta(1) ligand mimetic CS-1 peptide. Additionally, recognition of the MDC-L disintegrin domain required "activation" of lymphocyte beta(1) integrins. The interaction of MDC-L with alpha(4)beta(1) may potentially regulate metalloprotease function by targeting or sequestering the active protease on the cell surface. These results suggest a potential role for the lymphocyte ADAM, MDC-L, in the interaction of lymphocytes with alpha(4)beta(1)-expressing leukocytes.     

Two novel monoclonal antibodies to fibronectin that recognize the Hep II and CS-1 regions respectively: their differential effect on lymphocyte adhesion.

We have obtained two new mAbs to the carboxy-terminal region of fibronectin, namely P3D4 and P1F11, and have studied their binding sites and their ability to block lymphocyte adhesion to fibronectin. ELISA and Western blot analyses showed that P3D4 reacts with both fibronectin chains and both Hep II-containing fragments (58 kDa and 38 kDa). P1F11, raised against the synthetic peptide CS-1, reacted with the 38 kDa fragment and with a 190 kDa fragment derived from the A chain of fibronectin. P1F11 did not react with the 58 kDa fragment thus clearly establishing that 58 kDa comes from the B chain of fibronectin and lacks the CS-1 sequence. mAbs P3D4 and P1F11 were used to evaluate the contribution of the Hep II and CS-1 sites in cell attachment to fibronectin. P3D4 effectively inhibited B cell adhesion to 38 kDa, 58 kDa and fibronectin; P1F11 however produced only limited inhibition, suggesting that lymphocyte interaction with Hep II may modulate further binding to the CS-1 site.     

Heparin II domain of fibronectin uses alpha4beta1 integrin to control focal adhesion and stress fiber formation, independent of syndecan-4

Co-signaling events between integrins and cell surface proteoglycans play a critical role in the organization of the cytoskeleton and adhesion forces of cells. These processes, which appear to be responsible for maintaining intraocular pressure in the human eye, involve a novel cooperative co-signaling pathway between alpha5beta1 and alpha4beta1 integrins and are independent of heparan sulfate proteoglycans. Human trabecular meshwork cells isolated from the eye were plated on type III 7-10 repeats of fibronectin (alpha5beta1 ligand) in the absence or presence of the heparin (Hep) II domain of fibronectin. In the absence of the Hep II domain, cells had a bipolar morphology with few focal adhesions and stress fibers. The addition of the Hep II domain increased cell spreading and the numbers of focal adhesions and stress fibers. Cell spreading and stress fiber formation were not mediated by heparan sulfate proteoglycans because treatment with chlorate, heparinase, or soluble heparin did not prevent Hep II domain-mediated cell spreading. Cell spreading and stress fiber formation were mediated by alpha4beta1 integrin because soluble anti-alpha4 integrin antibodies inhibited Hep II domain-mediated cell spreading and soluble vascular cell adhesion molecule-1 (alpha4beta1 ligand)-induced cell spreading. This is the first demonstration of the Hep II domain mediating cell spreading and stress fiber formation through alpha4beta1 integrin. This novel pathway demonstrates a cooperative, rather than antagonistic, role between alpha5beta1 and alpha4beta1 integrins and suggests that interactions between the Hep II domain and alpha4beta1 integrin could modulate the strength of cytoskeleton-mediated processes in the trabecular meshwork of the human eye.     

Structure-function analysis of the human integrin VLA-4 (alpha 4/beta 1). Correlation of proteolytic alpha 4 peptides with alpha 4 epitopes and sites of ligand interaction.

The structure-function relationship of the human integrin VLA-4 (alpha 4/beta 1; CD49d/CD29), has been studied in the human B-cell line Ramos by immunochemical and functional analysis. Ramos cells expressed the 150-kDa non-proteolyzed form of the alpha 4 chain, which could be digested upon mild trypsin treatment to generate the 80- and 65-kDa proteolyzed forms, as well as alpha 4 polypeptides of 55 and 50 kDa. In addition, treatment of Ramos cells with high doses of pronase predominantly yielded the 55- and 50-kDa alpha 4 peptides. The trypsin-generated 80- and 65-kDa alpha 4 polypeptides, but not the 55- and 50-kDa fragments, were able to associate with the beta 1 chain. Distinct anti-VLA-4 mAb against four different alpha 4 epitopes, referred to as epitopes A, B1, B2, and C, recognized the 150-kDa alpha 4 chain both associated or non-associated with the beta 1 chain. The alpha 4 proteolytic forms of 80, 65 and 50 kDa were precipitated by the anti-alpha 4 mAb directed against the four different alpha 4 epitopes. On the other hand, the 55-kDa alpha 4 peptide was present in precipitates from anti-alpha 4 mAb specific for epitopes A, B1 and C, but absent in precipitates from the anti-alpha 4 mAb specific for epitope B2. The different adhesive capacities of the VLA-4 integrin, namely the interaction with a 38-kDa fibronectin fragment containing the CS-1 region of plasma fibronectin (Fn-38), the binding to the vascular cell adhesion molecule-1 (VCAM-1), or the ability to mediate the anti-alpha 4-induced cell aggregation, were not altered on VLA-4 from cells upon mild trypsin treatment, when compared to non-treated cells. However, the 55- and 50-kDa alpha 4 forms generated by high-dose pronase cell treatment, failed to mediate cell interaction with Fn-38 or VCAM-1 ligands, and cell aggregation could not be triggered through VLA-4 under these conditions.     

Distinct structural requirements for binding of the integrins alphavbeta6, alphavbeta3, alphavbeta5, alpha5beta1 and alpha9beta1 to osteopontin.

The extracellular matrix protein, osteopontin, is a ligand for several members of the integrin family, including alpha5beta1, alphavbeta3, alphavbeta5 and alpha9beta1. Osteopontin is a substrate for a number of extracellular proteases, including thrombin and the metalloproteases MMP-3 and MMP-7, which cleave osteopontin at sites close to or within the mapped integrin binding sites. Using affinity chromatography and cell adhesion assays, we now identify the integrin alphavbeta6 as an additional osteopontin receptor. Utilizing a series of recombinant forms of osteopontin, we compared the structural requirements for alphavbeta6 binding with those for the 4 other osteopontin-binding integrins. Like alpha5beta1, alphavbeta3 and alphavbeta5 (but not alpha9beta1), alphavbeta6 binds to the RGD site in osteopontin, since RGD peptide or mutation of this site to RAA completely inhibits alphavbeta6-mediated cell adhesion. For both alpha9beta1 and alpha5beta1, the N-terminal fragment generated by thrombin cleavage is a much better ligand than full length osteopontin, whereas thrombin-cleavage does not appear to be required for optimal adhesion to alphavbeta3, alphavbeta5 or alphavbeta6. A recombinant fragment predicted to be generated by MMP cleavage no longer supported alpha5beta1 or alpha9beta1-mediated adhesion, but adhesion mediated by alphavbeta5 or alphavbeta6 was unaffected. Finally, adhesion of alphavbeta5 or alphavbeta6 was inhibited by mutation of two aspartic acid residues upstream of the RGD site, whereas adhesion mediated by alphavbeta3, alpha5beta1 or alpha9beta1 was unaffected by these mutations. These results suggest that the hierarchy of integrin interactions with osteopontin can undergo complex regulation at least in part through the action of extracellular proteases.     

The properdin-like type I repeats of human thrombospondin contain a cell attachment site

Thrombospondin (TS) is a modular adhesive glycoprotein that contains three domains previously implicated in the attachment of cells to TS. These include the amino-terminal heparin-binding domain, the carboxy terminal cell or platelet-binding domain, and an RGDA sequence of TS. We have characterized a mAb against human TS, designated A4.1, which inhibits the attachment of human melanoma cells (G361) to TS. The epitope for A4.1 lies within the amino terminal half of the central stalklike region of TS which is distinct from the three known cell attachment sites. This region of TS is recovered in a 50-kD peptide after chymotryptic digestion of TS in EDTA. It contains the procollagen-like domain of TS as well as three type I repeats of a 60-residue segment homologous to two malarial proteins and the complement proteins properdin, and factors C6 through C9. The purified chymotryptic fragment is an effective attachment factor for G361 cells. A4.1 blocks adhesion to the 50-kD domain, as do some sulfated glycoconjugates. RGD (and RGE) peptides and mAbs against other domains of TS are not inhibitory. Peptides (19 mers) based on the core homology sequence of the three type I repeats of TS are potent attachment factors for these cells, and this adhesion is also inhibited by sulfated glycoconjugates. A polyclonal antibody raised against one of these peptides inhibits adhesion of G361 cells to the peptides, to the 50-kD fragment and to intact TS. Thus a new cell-adhesion site has been identified in TS whose sequence is very similar to the site identified in region II of the circumsporozoite protein of malaria parasites (Rich, K. A., F. W. George IV, J. L. Law, and W. J. Martin. 1990. Science (Wash. DC) 249:1574-1577. Thus there may be a common receptor which binds TS, malarial proteins, and properdin.     

Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis

Leukocyte emigration possibly requires dynamic regulation of integrin adhesiveness for endothelial and extracellular matrix ligands. Adhesion assays on purified vascular cell adhension molecule (VCAM)-1, fibronectin, and fibronectin fragments revealed distinct kinetic patterns for the regulation of very late antigen (VLA)-4 (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) avidity by the CC chemokines monocyte inflammatory protein (MIP)-1 alpha, RANTES (regulated on activation, normal T expressed and secreted), or monocyte chemoattractant protein (MCP)-1 in monocytes. CC chemokines induced early activation and subsequent deactivation of VLA-4, whereas upregulation of VLA-5 avidity occurred later and persisted. Controlled detachment assays in shear flow suggested that adhesive strength of VLA-4 for VCAM-1 or the 40-kD fragment of fibronectin (FN40) is more rapidly increased and subsequently reduced by MCP-1 than by MIP-1 alpha, and confirmed late and sustained activation of the adhesive strength of VLA-5 for the 120- kD fragment of fibronectin (FN120). Mn2+ or the stimulating beta 1 mAb TS2/16 strongly and stably enhanced monocyte binding to VCAM-1 or fibronectin, and locked beta 1 integrins in a high avidity state, which was not further modulated by CC chemokines. Mn2+ and mAb TS2/16 inhibited CC chemokine-induced transendothelial migration, particularly chemotaxis across stimulated endothelium that involved VLA-4 and VCAM- 1. VLA-4 on Jurkat cells is of constitutively high avidity and interfered with migration across barriers expressing VCAM-1. Low but not high site densities of VCAM-1 or FN40 promoted, while FN120 impaired, beta 1 integrin-dependent monocyte chemotaxis to MCP-1 across filters coated with these substrates. Thus, we show that CC chemokines can differentially and selectively regulate avidity of integrins sharing common beta subunits. Transient activation and deactivation of VLA-4 may serve to facilitate transendothelial diapedesis, whereas late and prolonged activation of VLA-5 may mediate subsequent interactions with the basement membrane and extracellular matrix.     

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.     

Activation-dependent recognition by hematopoietic cells of the LDV sequence in the V region of fibronectin

It has been shown that the alpha 4 beta 1 integrin is the lymphocyte receptor for the carboxy terminal cell-binding domain of fibronectin which comprises adhesion sites in Hep 2 and a high affinity site, CS-1, in the type III connecting segment or V (for variable) region. In the present studies, using a series of peptides derived from CS-1, we identify the tripeptide leu-asp-val (LDV), as the minimal peptide capable of supporting stable lymphocyte or melanoma cell adhesion. However, only cells which expressed an active form of the alpha 4 beta 1 complex were capable of attaching to and spreading on LDV peptide. On a molar basis, LDV minimal peptides were either not active or 10-20 times less active than intact CS-1 in promoting the adhesion of lymphocytes expressing the resting form of the receptor. In cells which express the high avidity form of the receptor, LDV and CS-1 were equally effective in promoting cell adhesion and spreading. The avidity of the alpha 4 beta 1 complex could be altered with mAbs to beta 1 which specifically activate beta 1 dependent function. The high avidity form of the alpha 4 beta 1 complex could be induced on U937 cells, T, and B lymphoblastoid cell lines, or PHA-stimulated T cell blasts. Resting PBL could not be induced to bind LDV peptide conjugates by activating antibodies to beta 1 implying that two signals are required for LDV recognition by T cells. In conclusion, these data show clearly that the minimal peptide for the alpha 4 beta 1 complex in CS-1 is the LDV sequence. Although numerous cell populations can interact with intact CS-1 only cells which express an active alpha 4 beta 1 complex can bind the LDV sequence. This implies that cell interaction with the carboxy terminal cell-binding domain of fibronectin can be regulated at several levels: (a) alpha 4 beta 1 expression; (b) activation of the alpha 4 beta 1 complex; and (c) alternate splicing of CS-1 into V+ isoforms of fibronectin.     

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.