ADAM15 suppresses cell motility by driving integrin alpha5beta1 cell surface expression via Erk inactivation

Human ADAM15 is unique among the A disintegrin and metalloprotease domain (ADAM) family because of the integrin binding motif Arg-Gly-Asp (RGD) within its disintegrin domain. Integrin alpha5beta1 has been reported to bind to ADAM15 in an RGD-dependent manner, but the biological significance of the interaction between ADAM15 and alpha5beta1 is unknown. To characterize the effects of ADAM15 on alpha5beta1-mediated cell adhesion and migration and elucidate the potential mechanism, CHO cells which express endogenous integrin alpha5beta1 were transfected with human ADAM15 cDNA. ADAM15 overexpression led to enhanced cell adhesion and decreased migration on fibronectin, which were suppressed by down-regulation of integrin alpha5. Overexpression of ADAM15 not only increased the cell surface expression of integrin alpha5 but also resulted in a more clustered staining of alpha5 on cell surface, while the beta1 subunit remained unchanged. Unexpectedly, results from immunoprecipitation and immunofluorescence indicated that ADAM15 and alpha5beta1 integrin did not interact directly in CHO cells. We found that ADAM15 expression decreased the phosphorylation of Erk1/2. Consistently, down-regulation of Erk1/2 phosphorylation by MEK inhibitor PD98059 or siRNA against Erk1/2 enhanced the expression of alpha5 on cell surface. By using a B16F10 pulmonary metastasis model, we revealed that overexpression of ADAM15 significantly reduced the number of metastatic nodules on the lung. Taken together, this study reveals for the first time that ADAM15 could drive alpha5 integrin expression on cell surface via down-regulation of phosphorylated Erk1/2. This presents a novel mechanism by which ADAM15 regulates cell-matrix adhesion and migration.     

Expression of chicken integrin beta 1 subunit in rat PC12 cells

We transfected rat pheochromocytoma (PC12) cells with a cDNA encoding chicken integrin beta 1 subunit. The chicken integrin beta 1 subunit produced in stable transfectants associated with two major alpha subunits of rat integrins to form interspecific chimeric receptors. These receptors mediated cell spreading and initial neurite outgrowth on laminin as did corresponding endogenous integrins, although they were slightly less effective in inducing cell adhesion to laminin. These results indicate that chicken integrin beta 1 may functionally substitute for beta 1 subunit of rat integrins in PC12 cells. Apparently, the structure of the integrin beta 1 subunit is highly conserved in the evolution of these species.     

The integrin alpha 7 cytoplasmic domain regulates cell migration, lamellipodia formation, and p130CAS/Crk coupling

The integrin alpha(7)beta(1) is the major laminin-binding integrin in skeletal, heart, and smooth muscle and is a receptor for laminin-1 and -2. It mediates myoblast migration on laminin-1 and -2 and thus might be involved in muscle development and repair. Previously we have shown that alpha(7)B as well as the alpha(7)A and -C splice variants induce cell motility on laminin when transfected into nonmotile HEK293 cells. In this study we have investigated the role of the cytoplasmic domain of alpha(7) in the laminin-induced signal transduction of alpha(7)beta(1) integrin regulating cell adhesion and migration. Deletion of the cytoplasmic domain did not affect assembly of the mutated alpha(7)Deltacyt/beta(1) heterodimer on the cell surface or adhesion of alpha(7)Deltacyt-transfected cells to laminin. The motility of these cells on the laminin-1/E8 fragment, however, was significantly reduced to the level of mock-transfected cells; lamellipodia formation and polarization of the cells were also impaired. Adhesion to the laminin-1/E8 fragment induced tyrosine phosphorylation of the focal adhesion kinase, paxillin, and p130(CAS) as well as the formation of a p130(CAS)-Crk complex in wild-type alpha(7)B-transfected cells. In alpha(7)BDeltacyt cells, however, the extent of p130(CAS) tyrosine formation was reduced and formation of the p130(CAS)-Crk complex was impaired, with unaltered levels of p130(CAS) and Crk protein levels. These findings indicate adhesion-dependent regulation of p130(CAS)/Crk complex formation by the cytoplasmic domain of alpha(7)B integrin after cell adhesion to laminin-1/E8 and imply alpha(7)B-controlled lamellipodia formation and cell migration through the p130(CAS)/Crk protein complex.     

The beta1 cytoplasmic domain regulates the laminin-binding specificity of the alpha7X1 integrin

During muscle development, the laminin-specific alpha7 integrin is alternatively spliced in the putative ligand-binding domain to yield either the alpha7X1 or the alpha7X2 variant. The relative level of alpha7X1 and alpha7X2 is developmentally regulated. Similarly, the partner beta1 integrin cytoplasmic domain is converted from the beta1A to the beta1D splice variant. To determine whether beta1D modulates the activity of the alpha7 receptor, cells were transfected with alpha7X1 and beta1D cDNA. alpha7X1 coupled with beta1A failed to adhere to laminin-1, whereas cotransfectants expressing alpha7X1 and beta1D showed strong adhesion. Interestingly, alpha7X1 complexed with beta1A and beta1D displayed the same level of poor adhesion to laminin-2/4 or strong adhesion to laminin-10/11. These findings indicate that alpha7 function is regulated not only by X1/X2 in its extracellular domain but also by beta1 cytoplasmic splice variants. It is likely that expression of beta1D alters alpha7X1 binding to laminin isoforms by a process related to ligand affinity modulation. Functional regulation of alpha7beta1 by developmentally regulated splicing events may be important during myogenic differentiation and repair because the integrin mediates adhesion, motility, and cell survival.     

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

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

Adhesion properties of human bladder cell lines with extracellular matrix components: the role of integrins and glycosylation

Integrin subunits present on human bladder cells displayed heterogeneous functional specificity in adhesion to extracellular matrix proteins (ECM). The non-malignant cell line (HCV29) showed significantly higher adhesion efficiency to collagen IV, laminin (LN) and fibronectin (FN) than cancer (T24, Hu456) and v-raf transfected (BC3726) cell lines. Specific antibodies to the alpha(2), alpha(5) and beta(1) integrin subunits inhibited adhesion of the non-malignant cells, indicating these integrin participation in the adhesion to ECM proteins. In contrast, adhesion of cancer cells was not inhibited by specific antibodies to the beta(1) integrin subunit. Antibodies to alpha(3) integrin increased adhesion of cancer cells to collagen, LN and FN, but also of the HCV29 line with collagen. It seems that alpha(3) subunit plays a major role in modulation of other integrin receptors especially in cancer cells. Differences in adhesion to ECM proteins between the non-malignant and cancer cell lines in response to Gal and Fuc were not evident, except for the v-raf transfected cell line which showed a distinct about 6-fold increased adhesion to LN on addition of both saccharides. N-Acetylneuraminic acid inhibited adhesion of all cell lines to LN and FN irrespective of their malignancy.     

Transfection of MCF-7 carcinoma cells with human integrin alpha7 cDNA promotes adhesion to laminin

The laminin-binding alpha7beta1 integrin receptor is highly expressed by skeletal and cardiac muscles, and has been suggested to be a crucial molecule during myogenic cell migration and differentiation. Absence of integrin alpha7 subunit contributes to a form of muscular dystrophy in integrin alpha7 null mice, whereas specific mutations in the alpha7 gene are associated in humans with congenital myopathy. To examine in more detail the potential role of integrin alpha7 in human-related muscular disorders, we cloned alpha7 cDNA by RT-PCR from human skeletal muscle mRNA and then expressed the full-length human integrin alpha7 cDNA by transfection in several cell lines including MCF-7, COS-7, and NIH3T3 cells. The isolated cDNA corresponds to the human alpha7X2B alternative splice form. Expression of human alpha7 was further confirmed by transfection of chimeric human/mouse alpha7 cDNA constructs. To demonstrate the functionality of expressed human alpha7, adhesion experiments with transfected MCF-7 cells have confirmed the specific binding of human alpha7 to laminin. In addition, mouse polyclonal and monoclonal antibodies were generated against the extracellular domain of human alpha7 and used to analyze by flow cytometry MCF-7 and NIH3T3 cells transfected with the full-length of human alpha7 cDNA. These results show for the first time the exogenous expression of functional full-length human alpha7 cDNA, as well as the development of monoclonal antibodies against the human alpha7 extracellular domain. Antibodies developed will be useful for further analysis of human disorders involving alpha7 dysfunction and facilitate isolation of muscle stem cells (satellite cells) and thereby expand the opportunities for genetically modified transplantation treatment of human disease.     

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.     

The muscle integrin binding protein (MIBP) interacts with alpha7beta1 integrin and regulates cell adhesion and laminin matrix deposition

Integrins are alphabeta transmembrane receptors that function in key cellular processes, including cell adhesion, differentiation, and extracellular matrix deposition through interactions with extracellular, membrane, and cytoplasmic proteins. We previously identified and cloned a muscle beta1 integrin cytoplasmic binding protein termed MIBP and found that the expression level of MIBP is critical in the decision-making process of terminal myogenic differentiation. We report here that MIBP interacts with the alpha7beta1 integrin but not the alpha5beta1 integrin in C2C12 myoblasts, suggesting an important role of integrin alpha chains in the regulation of the beta1-MIBP interaction. Furthermore, consistent with its selective binding activity toward the alpha7beta1 laminin receptor, we have found that overexpression of MIBP in C2C12 myoblasts resulted in a significant reduction of cell adhesion to laminin and inhibition of laminin matrix deposition. By contrast, neither cell adhesion to fibronectin nor fibronectin matrix deposition was significantly altered in cells overexpressing MIBP. Finally, we show that both the protein level and tyrosine phosphorylation of paxillin, a key signaling molecule involved in the cellular control of myogenic differentiation, are increased by MIBP. These results suggest that MIBP functions in the control of myogenic differentiation by regulating alpha7beta1 integrin-mediated cell interactions with laminin matrix and intracellular signaling through paxillin.     

The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading

The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP). Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking beta1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did not spread on ADAM 12. However, spreading could be efficiently induced by the addition of either 1 mM Mn(2+) or the beta1 integrin-activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12-syndecan complex fails to modulate the function of beta1 integrin.     

The role of extracellular and cytoplasmic splice domains of alpha7-integrin in cell adhesion and migration on laminins

The major laminin-binding integrin of skeletal, smooth, and heart muscle is alpha7beta1-integrin, which is structurally related to alpha6beta1. It occurs in three cytoplasmic splice variants (alpha7A, -B, and -C) and two extracellular forms (X1 and X2) which are developmentally regulated and differentially expressed in skeletal muscle. Previously, we have shown that ectopic expression of the alpha7beta-integrin splice variant in nonmotile HEK293 cells specifically induced cell locomotion on laminin-1 but not on fibronectin. To investigate the specificity and the mechanism of the alpha7-mediated cell motility, we expressed the three alpha7-chain cytoplasmic splice variants, as well as alpha6A- and alpha6B-integrin subunits in HEK293 cells. Here we show that all three alpha7 splice variants (containing the X2 domain), as well as alpha6A and alpha6B, promote cell attachment and stimulate cell motility on laminin-1 and its E8 fragment. Deletion of the cytoplasmic domain (excluding the GFFKR consensus sequence) from alpha7B resulted in a loss of the motility-enhancing effect. On laminin-2/4 (merosin), the predominant isoform in mature skeletal muscle, only alpha7-expressing cells showed enhanced motility, whereas cells transfected with alpha6A and alpha6B neither attached nor migrated on laminin-2. Adhesion of alpha7-expressing cells to both laminin-1 and laminin-2 was specifically inhibited by a new monoclonal antibody (6A11) specific for alpha7. Expression of the two extracellular splice variants alpha7X1 and alpha7X2 in HEK293 cells conferred different motilities on laminin isoforms: Whereas alpha7X2B promoted cell migration on both laminin-1 and laminin-2, alpha7X1B supported motility only on laminin-2 and not on laminin-1, although both X1 and X2 splice variants revealed similar adhesion rates to laminin-1 and -2. Fluorescence-activated cell sorter analysis revealed a dramatic reduction of surface expression of alpha6-integrin subunits after alpha7A or -B transfection; also, surface expression of alpha1-, alpha3-, and alpha5-integrins was significantly reduced. These results demonstrate selective responses of alpha6- and alpha7-integrins and of the alpha7 splice variants to laminin-1 and -2 and indicate differential roles in laminin-controlled cell adhesion and migration.     

Inhibitory effects of MLDG-containing heterodimeric disintegrins reveal distinct structural requirements for interaction of the integrin alpha 9beta 1 with VCAM-1, tenascin-C, and osteopontin

The integrin alpha9beta1 is expressed on epithelial cells, smooth muscle cells, skeletal muscle, and neutrophils and recognizes at least three distinct ligands: vascular cell adhesion molecule 1 (VCAM-1), tenascin-C, and osteopontin. The alpha9 subunit is structurally similar to the integrin alpha4 subunit, and alpha9beta1 and alpha4beta1 both recognize VCAM-1 as a ligand. We therefore examined whether the disintegrin EC3, which we have recently shown specifically inhibits the binding of alpha4 integrins to ligands, would also be a functional inhibitor of alpha9beta1. EC3 and a novel heterodimeric disintegrin that we identified, EC6, both were potent inhibitors of alpha9beta1-mediated adhesion to VCAM-1 and of neutrophil migration across tumor necrosis factor-activated endothelial cells. A peptide containing a novel MLDG motif shared by both of these disintegrins also inhibited alpha9beta1- and alpha4beta1-mediated adhesion to VCAM-1. Surprisingly though, concentrations of EC3 that completely inhibited adhesion of alpha9-transfected cells to VCAM-1 had little or no effect on adhesion to either of the other alpha9beta1 ligands, osteopontin and tenascin-C. Furthermore, peptides AEIDGIEL and SVVYGLR, which we have previously shown inhibit binding of alpha9beta1-expressing cells to tenascin-C and osteopontin, respectively, had no effect on adhesion to VCAM-1. These data suggest that there are structurally distinct requirements for interactions of the alpha9beta1 integrin with VCAM-1 and the extracellular matrix ligands osteopontin and tenascin-C.     

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.     

Activation of protein kinase C by phorbol esters modulates alpha2beta1 integrin on MCF-7 breast cancer cells

Cellular adhesions to other cells and to the extracellular matrix play crucial roles in the malignant progression of cancer. In this study, we investigated the role of protein kinase C (PKC) in the regulation of cell-substratum adhesion by the breast adenocarcinoma cell line MCF-7. A PKC activator, 12-O-tetradecanoylphorbol-l, 3-acetate (TPA), stimulated cell adhesion to laminin and collagen I in a dose-dependent manner over a 1- to 4-h interval. This enhanced adhesion was mediated by alpha2beta1 integrin, since both anti-alpha2 and anti-beta1 blocking antibodies each completely abrogated the TPA-induced adhesion. FACS analysis determined that TPA treatment does not change the cell surface expression of alpha2beta1 integrin over a 4-h time interval. However, alpha2beta1 levels were increased after 24 h of TPA treatment. Thus, the enhanced avidity of alpha2beta1-dependent cellular adhesion preceded the induction of alpha2beta1 cell surface expression. Northern blot analysis revealed that mRNA levels of both alpha2 and beta1 subunits were increased after exposure to TPA for 4 h, indicating that the induction of alpha2beta1 mRNA preceded that of its cell surface expression. This further suggested that the TPA-induced avidity of alpha2beta1 was independent of increased expression of alpha2beta1. Pretreatment of cells with the PKC inhibitor calphostin C partially antagonized the TPA-induced increase in expression of alpha2beta1 integrin expression and of alpha2beta1-mediated cellular adhesion. To identify a possible mechanism by which TPA could be acting to promote the rapid induction of alpha2beta1 adhesion, we treated the cells with the Rho-GTPase inhibitor Clostridium botulinumexotoxin C3. C3 inhibited TPA-induced adhesion to laminin and collagen I in a dose-dependant manner, suggesting a likely role for Rho in TPA-induced adhesion. Together, these results suggest that PKC can modulate the alpha2beta1-dependent adhesion of MCF-7 cells by two distinct mechanisms: altering the gene expression of integrins alpha2 and beta1 and altering the avidity of the alpha2beta1 integrin by a Rho-dependant mechanism.     

The laminin-binding activity of the alpha 7 integrin receptor is defined by developmentally regulated splicing in the extracellular domain.

The expression pattern of the laminin-binding alpha 7 beta 1 integrin is developmentally regulated in skeletal, cardiac, and smooth muscle. The X1/X2 alternative splicing in the extracellular domain of alpha 7 is found in the variable region between conserved alpha-chain homology repeat domains III and IV, a site implicated in ligand binding. To assess differences in X1/X2 isoform activity, we generated MCF-7 cell lines transfected with alpha 7-X1/X2 cDNAs. Transfectants expressing the alpha 7-X2 variant adhered rapidly to laminin 1, whereas those expressing alpha 7-X1 failed to attach. That alpha 7-X1 exists in an inactive state was established in assays using an activating beta 1 antibody that induced X1-dependent cell adhesion and spreading. Furthermore, the activation of alpha 7-X1 was cell type specific, and when expressed in HT1080 cells, the integrin was converted into a fully functional receptor capable of promoting adhesion. Thus, the expression of the alpha 7-X1/X2 integrin is a novel mechanism that regulates receptor affinity states in a cell-specific context and may modulate integrin-dependent events during muscle development and repair.     

Distinct and overlapping ligand specificities of the alpha 3A beta 1 and alpha 6A beta 1 integrins: recognition of laminin isoforms.

The ligand specificity of the alpha 3A beta 1 integrin was analyzed using K562 cells transfected with full-length alpha 3A cDNA and was compared with that of alpha 6A beta 1 in similarly transfected K562 cells. Clones were obtained that showed comparable surface expression of either alpha 3A beta 1 or alpha 6A beta 1 integrins. Those expressing alpha 3A beta 1 attached to and spread on immunopurified human kalinin and cellular matrices containing human kalinin, which is a particular isoform of laminin. In addition, alpha 3A transfectants adhered to bovine kidney laminins possessing a novel A chain variant. Binding to kalinin was blocked by a monoclonal antibody against the A chain constituent of kalinin and adhesion to both kalinin and kidney laminins by anti-alpha 3 and beta 1 monoclonal antibodies. The alpha 3A transfected cells bound more strongly to kalinin and bovine kidney laminins after treatment with the beta 1 stimulatory antibody TS2/16. A distinctly weaker and activation-dependent adhesion of alpha 3A transfectants was observed on human placental laminins possessing the Am chain variant (merosin), and no adhesion occurred on bovine heart laminins and murine EHS tumor laminin. Further inactive substrates were fibronectin, nidogen, and collagen types IV and VI, indicating that the alpha 3A beta 1 integrin is a much less promiscuous receptor than thought before. By contrast, alpha 6A transfected cells adhered to all laminin isoforms when stimulated with TS2/16. Adhesion also occurred only on bovine kidney laminins in the absence of TS2/16. These results demonstrate that both alpha 3A beta 1 and alpha 6A beta 1 integrins are typical laminin receptors but that their affinity and activation dependence for binding to various laminin isoforms differ considerably.     

Regulation of alpha 6 beta 1 integrin laminin receptor function by the cytoplasmic domain of the alpha 6 subunit

The alpha 6 beta 1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha 6 subunit cytoplasmic domain in alpha 6 beta 1 integrin activation was examined. The use of P388D1 cells, an alpha 6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha 6A or alpha 6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated alpha 6 cDNA, alpha 6-delta CYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric alpha 6-delta CYT beta 1 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca++ and Mg++. The alpha 6A-delta CYT transfectants adhered to laminin, however, when Ca++/Mg++ was replaced with 150 microM Mn++. We also assessed the role of serine phosphorylation in the regulation of alpha 6A beta 1 integrin function by site-directed mutagenesis of the two serine residues present in the alpha 6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of alpha 6 A beta 1. Point mutations were introduced in the alpha 6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D1 transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type alpha 6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the alpha 6A cytoplasmic domain is not involved in this regulation.     

ADAM12 and alpha9beta1 integrin are instrumental in human myogenic cell differentiation

Knowledge on molecular systems involved in myogenic precursor cell (mpc) fusion into myotubes is fragmentary. Previous studies have implicated the a disintegrin and metalloproteinase (ADAM) family in most mammalian cell fusion processes. ADAM12 is likely involved in fusion of murine mpc and human rhabdomyosarcoma cells, but it requires yet unknown molecular partners to launch myogenic cell fusion. ADAM12 was shown able to mediate cell-to-cell attachment through binding alpha9beta1 integrin. We report that normal human mpc express both ADAM12 and alpha9beta1 integrin during their differentiation. Expression of alpha9 parallels that of ADAM12 and culminates at time of fusion. alpha9 and ADAM12 coimmunoprecipitate and participate to mpc adhesion. Inhibition of ADAM12/alpha9beta1 integrin interplay, by either ADAM12 antisense oligonucleotides or blocking antibody to alpha9beta1, inhibited overall mpc fusion by 47-48%, with combination of both strategies increasing inhibition up to 62%. By contrast with blockade of vascular cell adhesion molecule-1/alpha4beta1, which also reduced fusion, exposure to ADAM12 antisense oligonucleotides or anti-alpha9beta1 antibody did not induce detachment of mpc from extracellular matrix, suggesting specific involvement of ADAM12-alpha9beta1 interaction in the fusion process. Evaluation of the fusion rate with regard to the size of myotubes showed that both ADAM12 antisense oligonucleotides and alpha9beta1 blockade inhibited more importantly formation of large (> or =5 nuclei) myotubes than that of small (2-4 nuclei) myotubes. We conclude that both ADAM12 and alpha9beta1 integrin are expressed during postnatal human myogenic differentiation and that their interaction is mainly operative in nascent myotube growth.     

Adhesion to vascular cell adhesion molecule 1 and fibronectin. Comparison of alpha 4 beta 1 (VLA-4) and alpha 4 beta 7 on the human B cell line JY.

Most mononuclear leukocytes and cell lines express the integrin alpha 4 beta 1 (VLA-4) heterodimer. In this study we have used Northern blotting and immunoprecipitation experiments to demonstrate that a B lymphoblastoid cell line (JY) expressed the integrin beta 7 subunit in association with alpha 4. These alpha 4 beta 7-positive JY cells bound poorly or not at all to VLA-4 ligands (soluble form of vascular cell adhesion molecule 1 (sVCAM-1) and the CS1 region of fibronectin). In contrast, a beta 1-positive variant of JY cells (selected to express a mixture of alpha 4 beta 1 and alpha 4 beta 7) bound avidly to VLA-4 ligands, and this binding was completely inhibitable by anti-alpha 4 and anti-beta 1 monoclonal antibodies. Thus, beta 1 expression appears to be a critically important component of VLA-4-mediated binding to its ligands. After either JY or JY-beta 1 cells were stimulated for 15 min with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, the majority of adhesion to VCAM or fibronectin remained alpha 4- and beta 1-dependent, but a low amount of adhesion to sVCAM-1 or fibronectin became alpha 4-dependent, beta 1-independent, thus suggesting a role for alpha 4 beta 7. In summary, we have found (i) that alpha 4 beta 7 makes little or no contribution to fibronectin or VCAM-1 binding on unstimulated JY cells, (ii) that alpha 4 beta 7 perhaps makes a minor contribution to ligand binding on 12-O-tetradecanoyl-phorbol-13-acetate-stimulated cells, and (iii) that alpha 4 beta 1 is the functionally dominant VCAM-1 and fibronectin receptor even when expressed in relatively low amounts compared to alpha 4 beta 7.