Human smooth muscle VLA-1 integrin: purification, substrate specificity, localization in aorta, and expression during development

A membrane glycoprotein complex was isolated and purified from human smooth muscle by detergent solubilization and affinity chromatography on collagen-Sepharose. The complex was identified as VLA-1 integrin and consisted of two subunits of 195 and 130 kD in SDS-PAGE. Liposomes containing the VLA-1 integrin adhered to surfaces coated with type I, II, III, and IV collagens, Clq subcomponent of the first component of the complement, and laminin. The liposomes specifically adhered to these proteins in a Ca2+, Mg2(+)-dependent manner, but did not bind to gelatin, fibronectin, and thrombospondin substrates. The expression of VLA-1 integrin in different human tissues and cell types, and during aorta smooth muscle development was studied by SDS-PAGE, and subsequent quantitative immunoblotting was performed with antibodies recognizing alpha 1 and beta 1 subunits of the VLA-1 integrin. A high level of VLA- 1 integrin expression was an exceptional feature of smooth muscles. Fibroblasts, endothelial cells, keratinocytes, striated muscles, and platelets contained trace amounts of VLA-1 integrin. In the 10-wk-old human fetal aorta, VLA-1 integrin was found only in smooth muscle cells whereas mesenchymal cells, surrounding aortic smooth muscle cells, were VLA-1 integrin negative. By the 24th wk of gestation, the amount of VLA- 1 integrin was significantly reduced in the aortic media (4.3-fold for alpha 1 subunit and 2.5-fold for beta 1 subunit) compared with that in the 10-wk-old aortic smooth muscle cells. After birth, the expression of VLA-1 integrin increased and in the 1.5-yr-old child aorta the VLA-1 integrin level was almost the same as in adult aortic media. Smooth muscle cells from intimal thickening of adult aorta express five times less alpha 1 subunit of VLA integrin that smooth muscle cells from adult aortic media. In primary culture of aortic smooth muscle cells, the content of the VLA-1 integrin was dramatically reduced and subcultured cells did not contain VLA-1 integrin at all.     

Multiple functional forms of the integrin VLA-2 can be derived from a single alpha 2 cDNA clone: interconversion of forms induced by an anti- beta 1 antibody

The integrin VLA-2 was previously found to bind to either collagen alone, or collagen plus laminin, but the mechanism for this cell- specific functional difference was unknown. Here we transfected VLA-2 alpha 2 subunit cDNA into K562 cells and obtained VLA-2 (called Form-O) which bound to neither collagen nor laminin. We then used a Matrigel selection procedure to enrich for a minor subpopulation of K562 cells stably expressing a form of VLA-2 (Form-C) that bound to collagen but not laminin. In contrast, the same alpha 2 cDNA transfected into RD cells yielded VLA-2 (Form-CL) which bound to both collagen and laminin. These Form-O, -C, and -CL activities were stably expressed during extended cell culture, and could not be qualitatively altered by adding phorbol esters or by exchaning the resident divalent cations. However, addition of stimulatory anti-beta 1 antibodies (TS2/16, A-1A5) rapidly converted VLA-2 Form-O and Form-C into Form-CL. Anti-beta 1 antibody stimulation of VLA-2 activity was observed not only on whole cells, but also with solubilized receptors. These results suggest (a) that the ligand binding specificity of VLA-2 can be determined by its cellular environment, rather than by variations in the primary sequence of the alpha 2 subunit, (b) that stably inactive or partly active VLA-2 can be rapidly converted to a fully active form through conformational changes initiated at a nonligand binding site on the beta 1 subunit, and (c) that the mechanisms for VLA-2 stimulation by phorbol ester and by antibody are quite distinct, because the latter does not require an intact cell.     

The I Domain is Essential for Echovirus 1 Interaction with VLA-2

VLA-2, the α2β1 integrin, mediates cell adhesion to collagen and laminin, and is the receptor for the human pathogen echovirus 1. Because of its similarity to domains present in other proteins that interact with collagen, a 191 amino acid region within the α2 subunit (the I domain) has been proposed as a potential site for ligand interactions. Although the α2 subunits of human and murine VLA-2 are 84% identical, human α2 promotes virus binding whereas murine α2 does not. We used murine/human chimeric α2 molecules to identify regions of the human molecule essential for virus binding. Virus bound efficiently to a chimeric protein in which the human I domain was inserted into murine α2, indicating that the human I domain is responsible for specific virus interactions. Monoclonal antibodies that inhibited virus attachment all recognized epitopes within the human I do-main, further suggesting that virus interacts with this portion of the molecule. Similarly, antibodies that prevented VLA-2-mediated cell adhesion to collagen also mapped to the I domain. These results indicate that the I domain plays a role in VLA-2 interactions both with virus and with extracellular matrix ligands.     

Isolation and characterization of EMS16, a C-lectin type protein from Echis multisquamatus venom, a potent and selective inhibitor of the alpha2beta1 integrin

We have isolated and characterized EMS16, a potent and selective inhibitor of the alpha2beta1 integrin, from Echis multisquamatus venom. It belongs to the family of C-lectin type of proteins (CLPs), and its amino acid sequence is homologous with other members of this protein family occurring in snake venoms. EMS16 (M(r) approximately 33K) is a heterodimer composed of two distinct subunits linked by S-S bonds. K562 cells transfected with alpha2 integrin selectively adhere to immobilized EMS16, but not to two other snake venom-derived CLPs, echicetin and alboaggregin B. EMS16 inhibits adhesion of alpha2beta1-expressing cells to immobilized collagen I at picomolar concentrations, and the platelet/collagen I interaction in solution at nanomolar concentrations. EMS16 inhibits binding of isolated, recombinant I domain of alpha2 integrin to collagen in an ELISA assay, but not the interaction of isolated I domain of alpha1 integrin with collagen IV. Studies with monoclonal antibodies suggested that EMS16 binds to the alpha2 subunit of the integrin. EMS16 inhibits collagen-induced platelet aggregation, but has no effect on aggregation induced by other agonists such as ADP, thromboxane analogue (U46619), TRAP, or convulxin. EMS16 also inhibits collagen-induced, but not convulxin-induced, platelet cytosolic Ca(2+) mobilization. In addition, EMS16 inhibits HUVEC migration in collagen I gel. In conclusion, we report a new, potent viper venom-derived inhibitor of alpha2beta1 integrin, which does not belong to the disintegrin family.     

Identification of integrin collagen receptors on human melanoma cells

Integrin receptors may mediate the adhesion of cells to a number of extracellular matrix components. We found that the attachment of human melanoma cells to collagen types I and IV was blocked by antibodies to the integrin beta 1 subunit but not by peptides containing the Arg-Gly-Asp sequence. Ligand affinity chromatography was used to search for integrin-related receptors which mediate adhesion to native collagens. Detergent extracts of surface 125I-iodinated melanoma cells were chromatographed on type I or IV collagen-Sepharose columns. Bound material was eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. EDTA, but not Arg-Gly-Asp peptides, eluted a mixture of two integrin-related heterodimeric complexes. Each complex contained the integrin beta 1 chain with Mr of 110,000 and a distinct alpha chain with Mr of either 200,000 or 150,000. Immunoprecipitation with specific monoclonal antibodies identified the complexes as very late activation antigen (VLA)-1 (alpha 1 beta 1) and VLA-2 (alpha 2 beta 1), respectively. The binding of these receptors to collagen appeared to be specific because they failed to be retained on fibronectin- or laminin-Sepharose columns. Immunofluorescent staining of cells on collagen substrates with antibodies to VLA-1 and VLA-2 localized these complexes in vinculin-positive adhesion plaques. In contrast, the receptor complexes were not detected in adhesion plaques of cells attached to fibronectin- or laminin-coated substrates. These results indicate that melanoma cells express at least two different integrin-related collagen-binding receptor complexes that appear to mediate cell adhesion to collagen.     

Subunit structure of a laminin-binding integrin and localization of its binding site on laminin

A laminin receptor was isolated from human MG-63 osteosarcoma cells by affinity chromatography on human laminin. The isolated receptor was defined as the alpha 3 beta 1 integrin by immunoprecipitation with subunit-specific antibodies. A previously unclassified laminin-binding integrin from rat cells was shown also to contain the alpha 3 subunit. Both receptors bound to human and mouse laminin in a radioreceptor assay. They also both bound to some extent to fibronectin in this assay, but only the MG-63 cell receptor showed binding to type IV collagen. The binding of the radiolabeled receptor to insoluble laminin was inhibited by unlabeled receptor, by soluble laminin, and by chymotryptic fragments of laminin that have previously been shown to contain neurite-promoting and cell attachment-promoting activities. Moreover, the receptor binding was also inhibited by monoclonal antibodies capable of inhibiting the neurite-promoting activity of laminin and known to bind to laminin near the junction of the long arm and its terminal globule. One of these antibodies was reactive with fusion proteins expressed from laminin cDNA clones. The immunoreactive clones corresponded to the COOH-terminal end of the B1 subunit. These results identify the integrin-type laminin receptor isolated from the osteosarcoma cells as the alpha 3 beta 1 integrin and localize its binding site in close proximity of the B1 subunit COOH terminus.     

Endothelial cells use alpha 2 beta 1 integrin as a laminin receptor

Human umbilical vein endothelial cells attach and spread on laminin-coated substrates. Affinity chromatography was used to identify the attachment receptor. Fractionation of extracts from surface-iodinated endothelial cells on human laminin-Sepharose yielded a heterodimeric complex, the subunits of which migrated with molecular sizes corresponding to 160/120 kD and 160/140 kD under nonreducing and reducing conditions, respectively. The purified receptor bound to laminin and slightly less to fibronectin and type IV collagen in a radioreceptor assay. This endothelial cell laminin receptor was classified as an alpha 2 beta 1 integrin because monoclonal and polyclonal antibodies directed against the alpha 2 and bet 1 subunits immunoprecipitated the receptor. Cytofluorometric analysis and immunoprecipitation showed that the alpha 2 subunit is an abundant integrin alpha subunit in the endothelial cells and that the alpha subunits associated with laminin binding in other types of cells are expressed in these cells only at low levels. The alpha 2 beta 1 integrin appears to be a major receptor for laminin in the endothelial cells, because an anti-alpha 2 monoclonal antibody inhibited the attachment of the endothelial cells to human laminin. These results define a new role for the alpha 2 subunit in laminin binding and suggest that the ligand specificity of the alpha 2 beta 1 integrin, which is known as a collagen receptor in other types of cells, can be modulated by cell type-specific factors to include laminin binding.     

An abundant chick gizzard integrin is the avian alpha 1 beta 1 integrin heterodimer and functions as a divalent cation-dependent collagen IV receptor.

The alpha-subunit of an abundant chick gizzard integrin was isolated (T. Kelly, L. Molony, and K. Burridge, 1987, J. Biol. Chem. 262, 17,189-17,199) and fragmented by proteolytic digestion. The N-terminal sequences of the intact polypeptide and of several internal peptides were determined and were found to be highly homologous to the mammalian integrin alpha 1-subunit. Monoclonal antibodies to the chick integrin beta 1-chain react on immunoblots with the gizzard integrin beta-subunit (U. Hofer, J. Syfrig, and R. Chiquet-Ehrismann, 1990, J. Biol. Chem. 265, 14,561-14,565). The chain composition of the abundant chick gizzard integrin is therefore alpha 1 beta 1. Polyclonal antibodies to the avian integrin alpha 1-subunit block attachment of embryonic gizzard cells to human and chick collagen IV completely and inhibit attachment to mouse Engelbreth-Holm-Swarm (EHS) tumor laminin partially. In ELISA-style receptor assays, the isolated alpha 1 beta 1 integrin bound to human and chick collagen IV and to mouse EHS tumor and chick heart laminin. While the binding to collagen IV was abolished by removal of divalent cations, the binding to laminin was not sensitive to EDTA under the conditions used. Collagen I bound the isolated avian alpha 1 beta 1 integrin only weakly. As collagen IV was the only extracellular matrix protein for which a consistent, divalent cation-dependent, binding to the avian alpha 1 beta 1 integrin could be demonstrated in both cellular and molecular assays we suggest that it is a preferred ligand for this integrin.     

Molecular cloning of the rat integrin alpha 1-subunit: a receptor for laminin and collagen

Integrin heterodimers mediate a variety of adhesive interactions, including neuronal attachment to and process outgrowth on laminin. We report here the cloning and primary sequence of an M-200 kD integrin alpha subunit that associates with the integrin beta 1 subunit to form a receptor for both laminin and collagen. Similarities in ligand-binding specificity, relative molecular mass and NH2-terminal sequence make this a strong candidate for the rat homologue of the alpha subunit of the human integrin VLA-1. The full-length rat alpha 1 cDNAs encode a protein containing a purative signal sequence and a mature polypeptide of 1,152 amino acids, with extracellular, transmembrane and cytoplasmic domains. Several structural features are conserved with other integrin alpha chains, including (a) a sequence motif repeated seven times in the NH2-terminal half; (b) potential Ca2+/Mg2+ binding sites in repeats 5, 6, and 7, and (c) alignment of at least 14 of 23 cysteine residues. This rat alpha 1 sequence also contains a 206-amino acid I domain, inserted between repeats 2 and 3, that is homologous to I domains found in the same position in the alpha subunits of several integrins (VLA-2, Mac-1, LFA-1, p150). The rat alpha 1 and human VLA-2 apha subunits share greater than 50% sequence identity in the seven repeats and I domain, suggesting that these sequence identities may underlie some of their similar ligand-binding specificities. However, the rat integrin alpha 1 subunit has several unique features, including a 38-residue insert between two Ca2+/Mg2+ binding domains, and a divergent 15-residue cytoplasmic sequence, that may potentially account for unique functions of this integrin.     

Multiple cell surface receptors for the short arms of laminin: alpha 1 beta 1 integrin and RGD-dependent proteins mediate cell attachment only to domains III in murine tumor laminin

Cell surface molecules that interact with the cross formed by the three short arms of murine tumor laminin were studied using thermal perturbation, antibody and peptide blocking, and affinity chromatography. Several potential receptors for the laminin short arms were revealed that differed from those mediating cell attachment to the E8 (long arm) fragment. Two cell lines, Rugli and L8 attached well to E1-X (short arm) fragments of laminin. This attachment was blocked by antibodies against alpha 1 integrin chains. Other cells were unable to attach strongly to E1-X, but attached to P1. This attachment was unaffected by anti-beta 1 integrin antibodies, but specifically blocked by the peptide GRGDS. By contrast, binding of Rugli cells was RGD independent and blocked by anti-beta 1 integrin antibodies. G7 and C2C12 myoblasts were very sensitive to GRGDS (ID50 approximately 2 micrograms.ml-1) for attachment to P1 which implied that a non-beta 1 series integrin, possibly alpha v beta 3, was involved. On heat denaturation of P1(3) attachment remained sensitive to RGDS and ID50 was unchanged. On heat denaturation of E1-X, attachment remained sensitive to RGDS but the ID50 increased to approximately 200 micrograms.ml-1. Cellular beta 1 integrins were retained on laminin affinity columns. A beta 1 integrin with an approximately 190 kD alpha-chain could be isolated from Rugli cells whose attachment could be blocked by anti-alpha 1 antibodies and not from cells blocked by RGDS peptides. Anti-alpha 1 antibodies blocked Rugli attachment to native laminin, but only when the E8 cell binding sites on laminin were also blocked. Thus, a receptor related to alpha 1 beta 1 integrin can function simultaneously with a receptor for E8. Anti-alpha 1 also blocked attachment to heated laminin, suggesting that the heat-stable attachment activity in laminin involved the E1-X binding site. Thus, at least two putative receptors mediate attachment to the short arms of laminin. One, related to alpha 1 beta 1 integrin, recognizes RGDS-independent sites in E1-X defined by P1 (within domains III, IIIa, IIIb), and one is an RGD-dependent molecule recognizing sites in P1, and is not a beta 1 integrin.     

Human microvascular endothelial cells use beta 1 and beta 3 integrin receptor complexes to attach to laminin

Microvascular endothelial cells (MEC) use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured human MEC interact with laminin-rich basement membranes. By using a panel of monoclonal antibodies, we found that MEC cells express a number of integrin-related receptor complexes, including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha V beta 3. Attachment to laminin, a major adhesive protein in basement membranes, was studied in detail. Blocking monoclonal antibodies specific to different integrin receptor complexes showed that the alpha 6 beta 1 complex was important for MEC adhesion to laminin. In addition, blocking antibody also implicated the vitronectin receptor (alpha V beta 3) in laminin adhesion. We used ligand affinity chromatography of detergent-solubilized receptor complexes to further define receptor specificity. On laminin-Sepharose columns, we identified several integrin receptor complexes whose affinity for the ligand was dependent on the type of divalent cation present. Several beta 1 complexes, including alpha 1 beta 1, alpha 2 beta 1, and alpha 6 beta 1 bound strongly to laminin. In agreement with the antibody blocking experiments, alpha V beta 3 was found to bind well to laminin. However, unlike binding to its other ligands (e.g., vitronectin, fibrinogen, von Willebrand factor), alpha V beta 3 interaction with laminin did not appear to be Arg-Gly-Asp (RGD) sensitive. Finally, immunofluorescent staining demonstrated both beta 1 and beta 3 complexes in vinculin-positive focal adhesion plaques on the basal surface of MEC adhering to laminin-coated substrates. The results indicate that both these subfamilies of integrin heterodimers are involved in promoting MEC adhesion to laminin and the vascular basement membrane.     

Human colon carcinoma cells use multiple receptors to adhere to laminin: involvement of alpha 6 beta 4 and alpha 2 beta 1 integrins.

In this study, we used clone A, a human colon carcinoma cell line, to characterize those integrins that mediate colon carcinoma adhesion to laminin. Monoclonal antibodies specific for the human beta 1 subunit inhibited clone A adhesion to laminin. They also precipitated a complex of surface proteins that exhibited an electrophoretic behavior characteristic of alpha 2 beta 1 and alpha 3 beta 1. A monoclonal antibody specific for alpha 2 (PIH5) blocked clone A adhesion to laminin, as well as to collagen I. An alpha 3-specific antibody (P1B5) had no effect on clone A adhesion to laminin, even though it can block the adhesion of other cell types to laminin. Thus, the alpha 2 beta 1 integrin can function as both a laminin and collagen I receptor on clone A cells. Although these cells express alpha 3 beta 1, an established laminin receptor, they do not appear to use it to mediate laminin adhesion. In addition, the monoclonal antibody GoH3, which recognizes the alpha 6 integrin subunit, also inhibited carcinoma adhesion to laminin but not to fibronectin or collagen I. This antibody precipitated the alpha 6 subunit in association with the beta 4 subunit. There was no evidence of alpha 6 beta 1 association on these cells. In summary, the results obtained in this study indicate that multiple integrin alpha subunits, in association with two distinct beta subunits, are involved in colon carcinoma adhesion to laminin. Based on the behavior of alpha 3 beta 1 and alpha 2 beta 1, the results also suggest that cells can regulate the ability of a specific integrin to mediate adhesion.     

Identification of a tetrapeptide recognition sequence for the alpha 2 beta 1 integrin in collagen

The alpha 2 beta 1 integrin serves as either a specific cell surface receptor for collagen or as both a collagen and laminin receptor depending upon the cell type. Recently we established that the alpha 2 beta 1 integrin binds to a site within the alpha 1 (I)-CB3 fragment of type I collagen (Staatz, W. D., Walsh, J. J., Pexton, T., and Santoro, S. A. (1990) J. Biol. Chem. 265, 4778-4781). To define the alpha 2 beta 1 recognition sequence further we have prepared an overlapping set of synthetic peptides which completely spans the 148-amino acid alpha 1(I)-CB3 fragment and tested the peptides for ability to inhibit cell adhesion to collagen and laminin substrates. The minimal active recognition sequence defined by these experiments is a tetrapeptide of the sequence Asp-Gly-Glu-Ala (DGEA) corresponding to residues 435-438 of the type I collagen sequence. The DGEA-containing peptides effectively inhibited alpha 2 beta 1-mediated Mg2(+)-dependent adhesion of platelets, which use the alpha 2 beta 1 integrin as a collagen-specific receptor, to collagen but had no effect on alpha 5 beta 1-mediated platelet adhesion to fibronectin or alpha 6 beta 1-mediated platelet adhesion to laminin. In contrast, with T47D breast adenocarcinoma cells, which use alpha 2 beta 1 as a collagen/lamin receptor, adhesion to both collagen and laminin was inhibited by DGEA-containing peptides. Deletion of the alanine residue or substitution of alanine for either the glutamic or aspartic acid residues in DGEA-containing peptides resulted in marked loss of inhibitory activity. These results indicate that the amino acid sequence DGEA serves as a recognition site for the alpha 2 beta 1 integrin complex on platelets and other cells.     

Extracellular matrix receptors, ECMRII and ECMRI, for collagen and fibronectin correspond to VLA-2 and VLA-3 in the VLA family of heterodimers

The Very Late Activation Antigen (VLA) proteins are a family of five related heterodimers, which also are part of the integrin superfamily of cell adhesion molecules. Except for the identification of VLA-5 as a fibronectin receptor structure, the functions of the VLA proteins have remained unclarified. In this paper, immunoprecipitation experiments with both anti-alpha and anti-beta subunit antibodies showed that the previously identified cell adhesion receptor for collagen, extracellular matrix receptor II (ECMRII), is equivalent to VLA-2. At the same time a previously described multispecific cell adhesion receptor for collagen, fibronectin, and laminin (ECMRI) has been shown to be identical to VLA-3. Although the mAb 12F1 and P1H5 both recognized VLA-2 (ECMRII), they appeared to define distinct epitopes on the alpha 2 subunit. On the other hand, the mAb P1B5 and J143 recognized the alpha 3 subunit of VLA-3 (ECMRI) at or near the same site. Consistent with the collagen receptor functions of VLA-2 (ECMRII) and VLA-3 (ECMRI), anti-VLA beta antiserum blocked cell attachment to collagen.     

alpha 2beta 1 integrin is not recognized by rhodocytin but is the specific, high affinity target of rhodocetin, an RGD-independent disintegrin and potent inhibitor of cell adhesion to collagen

We have recombinantly expressed a soluble form of human alpha(2)beta(1) integrin that lacks the membrane-anchoring transmembrane domains as well as the cytoplasmic tails of both integrin subunits. This soluble alpha(2)beta(1) integrin binds to its collagen ligands the same way as the wild-type alpha(2)beta(1) integrin. Furthermore, like the wild-type form, it can be activated by manganese ions and an integrin-activating antibody. However, it does not bind to rhodocytin, a postulated agonist of alpha(2)beta(1) integrin from the snake venom of Calloselasma rhodostoma, which elicits platelet aggregation. Taking advantage of the recombinantly expressed, soluble alpha(2)beta(1) integrin, an inhibition assay was established in which samples can be tested for their capability to inhibit binding of soluble alpha(2)beta(1) integrin to immobilized collagen. Thus, by scrutinizing the C. rhodostoma snake venom in this protein-protein interaction assay, we found a component of the snake venom that inhibits the interaction of soluble alpha(2)beta(1) integrin to type I collagen efficiently. N-terminal sequences identified this inhibitor as rhodocetin, a recently published antagonist of collagen-induced platelet aggregation. We could demonstrate that its inhibitory effect bases on its strong and specific binding to alpha(2)beta(1) integrin, proving that rhodocetin is a disintegrin. Standing apart from the growing group of RGD-dependent snake venom disintegrins, rhodocetin interacts with alpha(2)beta(1) integrin in an RGD-independent manner. Furthermore, its native conformation, which is stabilized by disulfide bridges, is indispensibly required for its inhibitory activity. Rhodocetin does not contain any major collagenous structure despite its high affinity to alpha(2)beta(1) integrin, which binds to collagenous molecules much more avidly than to noncollagenous ligands, such as laminin. Blocking alpha(2)beta(1) integrin as the major collagen receptor on platelets, rhodocetin is responsible for hampering collagen-induced, alpha(2)beta(1) integrin-mediated platelet activation, leading to hemorrhages and bleeding disorders of the snakebite victim. Moreover, having a widespread tissue distribution, alpha(2)beta(1) integrin also mediates cell adhesion, spreading, and migration. We showed that rhodocetin is able to inhibit alpha(2)beta(1) integrin-mediated adhesion of fibrosarcoma cells to type I collagen completely.     

Regulation of the VLA integrin-ligand interactions through the beta 1 subunit

Integrins from the very late activation antigen (VLA) subfamily are involved in cellular attachment to extracellular matrix (ECM) proteins and in intercellular adhesions. It is known that the interaction of integrin proteins with their ligands can be regulated during cellular activation. We have investigated the regulation of different VLA-mediated adhesive interactions through the common beta 1 chain. We have found that certain anti-beta 1 antibodies strongly enhance binding of myelomonocytic U-937 cells to fibronectin. This beta 1-mediated regulatory effect involved both VLA-4 and VLA-5 fibronectin receptors. Moreover, anti-beta 1 mAb also induced VLA-4-mediated binding to a recombinant soluble form of its endothelial cell ligand VCAM-1. Non-activated peripheral blood T lymphocytes, unable to mediate VLA-4 interactions with fibronectin or VCAM-1, acquired the ability to bind these ligands in the presence of anti-beta 1 mAb. The anti-beta 1-mediated changes in the affinities of beta 1 integrin for their ligands were comparable to those triggered by different lymphocyte activation agents such as anti-CD3 mAb or phorbol ester. Adhesion of melanoma cells to other ECM proteins such as laminin or collagen as well as that of alpha 2-transfected K-562 cells to collagen, was also strongly enhanced by anti-beta 1 mAb. These beta 1-mediated regulatory effects on different VLA-ligand interactions do not involve changes in cell surface membrane expression of different VLA heterodimers. The anti-beta 1-mediated functional effects required an active metabolism, cytoskeleton integrity and the existence of physiological levels of intracellular calcium as well as a functional Na+/H+ antiporter. Beta 1 antibodies not only increased cell attachment but also promoted spreading and cytoplasmic extension of endothelial cells on plates coated with either fibronectin, collagen, or laminin as well as induced the rapid appearance of microspikes in U-937 cells on fibronectin. Moreover, both beta 1 integrin and the cytoskeletal protein talin colocalized in the anti-beta 1 induced microspikes. These results emphasize the central role of the common beta 1 chain in regulating different adhesive functions mediated by VLA integrins as well as cellular morphology.     

Deposition of laminin 5 by keratinocytes regulates integrin adhesion and signaling

Deposition of laminin 5 over exposed dermal collagen in epidermal wounds is an early event in repair of the basement membrane. We report that deposition of laminin 5 onto collagen switches adhesion and signaling from collagen-dependent to laminin 5-dependent. Ligation of laminin 5 by integrin alpha(6)beta(4) activates phosphoinositide 3-OH-kinase (PI3K) signaling. This activation allows for adhesion and spreading via integrin alpha(3)beta(1) on laminin 5 independent of RhoGTPase, a regulator of actin stress fibers. In contrast, adhesion and spreading on collagen via alpha(2)beta(1) is Rho-dependent and is inhibited by toxin B, a Rho inhibitor. Deposition of laminin 5 and ligation of alpha(6)beta(4) increases PI3K-dependent production of phosphoinositide di- and triphosphates, PI3K activity, and phosphorylation of downstream target protein c-Jun NH(2)-terminal kinase. Conversely, blocking laminin 5-deposition with brefeldin A, an inhibitor of vesicle transport, or with anti-laminin 5 monoclonal antibodies abolishes the PI3K-dependent spreading mediated by alpha(3)beta(1) and phosphorylation of c-Jun NH(2)-terminal kinase. Studies with keratinocytes lacking alpha(6)beta(4) or laminin 5 confirm that deposition of laminin 5 and ligation by alpha(6)beta(4) are required for PI3K-dependent spreading via alpha(3)beta(1). We suggest that deposition of laminin 5 onto the collagen substratum, as in wound repair, enables human foreskin keratinocytes to interact via alpha(6)beta(4) and to switch from a RhoGTPase-dependent adhesion on collagen to a PI3K-dependent adhesion and spreading mediated by integrin alpha(3)beta(1) on laminin 5.     

Human microvascular endothelial cells express integrin-related complexes that mediate adhesion to the extracellular matrix

Microvascular endothelial cells (MEC) must use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured MEC isolated from human foreskin interact with their subendothelial matrix. MEC were able to attach to diverse extracellular matrix proteins, including fibronectin (Fn), vitronectin (Vn), laminin (Ln), type I and IV collagen, as well as to fibrinogen and gelatin. Adhesion to Fn, but not to laminin or collagens, was specifically blocked in the presence of Arg-Gly-Asp (RGD)-containing peptides. When surface radioiodinated MEC were solubilized and subjected to affinity chromatography on Fn-Sepharose columns, two polypeptides of 150 and 125 kD, corresponding to the integrin heterodimer alpha 5 beta 1, were identified. MEC also express a complex of 150 (alpha) and 95 kD (beta 3) that is related to the Vn receptor. Immunofluorescent staining of MEC cultures with antibodies to the integrin beta 1 subunit demonstrated receptors on the basolateral surface at focal adhesion plaques that co-localized with vinculin and with Fn-positive matrix fibers. Occasionally, antibodies to the Vn receptor stained the vinculin-positive focal adhesion plaques that frequently co-localized with the beta 1 complex. However, in cultures of MEC that were attached to substrates coated with alternating strips of Fn and Vn, the beta 1 complex was preferentially localized to the Fn substrate, while the Vn receptor was concentrated on the Vn substrate. The results indicate that MEC express at least two different heterodimer adhesion receptors that belong to the integrin super-family and appear to have distinct ligand specificities: the Fn receptor and the Vn receptor. These receptors mediate cell adhesion to the extracellular matrix and presumably have an important role in hemostasis and neovascularization.     

Characterization of a laminin receptor on rat hepatocytes

The interaction of rat hepatocytes with laminin was studied. The cells were found to adhere to the distal half of the long arm in the laminin molecule (fragment E8), in addition to the previously identified site in the central cross of laminin (fragment P1). Attachment to laminin and to each of the two cell-binding fragments was inhibited by antibodies against the integrin beta 1-subunit of the fibronectin receptor, but not by the cell-binding peptide of fibronectin (Gly-Arg-Gly-Asp-Ser-Cys). By affinity chromatography on laminin-Sepharose in the presence of 2 mM Mn2+, the beta 1-subunit was isolated together with an alpha-subunit with an unreduced Mr of 180,000. This laminin-binding integrin did not bind to Sepharose conjugated with a 105-kDa cell-binding fragment of fibronectin and conversely, the fibronectin receptor of the cells (integrin alpha 5 beta 1) did not bind to the laminin-Sepharose. The 180-kDa protein was identified as the integrin subunit alpha 1 based on its specific reactivity with antibodies raised against a peptide of the N-terminal part of human alpha 1. Integrin alpha 1 beta 1 was found to bind at physiological ionic strength also to Sepharose conjugated with either one of the laminin fragments P1 or E8. Furthermore, integrin alpha 1 beta 1 isolated on one of the fragment columns could be shown to rebind to the other fragment-Sepharose. The results indicate that two structurally distinct domains of laminin may interact with the same type of receptor on hepatocytes.     

Crystal structure of the alpha1beta1 integrin I domain in complex with an antibody Fab fragment

The alpha1beta1 (VLA-1) integrin is a cell-surface receptor for collagen and laminin and has been implicated in biological pathways involved in several pathological processes. These processes may be inhibited by the monoclonal antibody AQC2, which binds with high affinity to human alpha1beta1 integrin. To understand the structural basis of the inhibition we determined the crystal structure of the complex of a chimeric rat/human I domain of the alpha1beta1 integrin and the Fab fragment of humanized AQC2 antibody. The structure of the complex shows that the antibody blocks the collagen binding site of the I domain. An aspartate residue, from the CDR3 loop of the antibody heavy chain, coordinates the MIDAS metal ion in a manner similar to that of a glutamate residue from collagen. Substitution of the aspartate residue by alanine or arginine results in significant reduction of antibody binding affinity. Interestingly, although the mode of metal ion coordination resembles that of the open conformation, the I domain maintains an overall closed conformation previously observed only for unliganded I domains.