Recognition of the laminin E8 cell-binding site by an integrin possessing the alpha 6 subunit is essential for epithelial polarization in developing kidney tubules

It has been previously shown that A-chain and domain(E8)-specific antibodies to laminin that inhibit cell adhesion also interfere with the establishment of epithelial cell polarity during kidney tubule development (Klein, G., M. Langegger, R. Timpl, and P. Ekblom. 1988. Cell. 55:331-341). A monoclonal antibody specific for the integrin alpha 6 subunit, which selectively blocks cell binding to E8, was used to study the receptors involved. Immunofluorescence staining of embryonic kidneys and of organ cultures of metanephric mesenchyme demonstrated coappearance of the integrin alpha 6 subunit and the laminin A-chain in regions where nonpolarized mesenchymal cells convert into polarized epithelial cells. Both epitopes showed marked colocalization in basal areas of tubules, while an exclusive immunostaining for alpha 6 was observed in lateral and apical cell surfaces of the tubular epithelial cells. Organ culture studies demonstrated a consistent inhibition of kidney epithelium development by antibodies against the alpha 6 subunit. The data suggest that the recognition of E8 cell-binding site of laminin by a specific integrin is crucial for the formation of kidney tubule epithelium from undifferentiated mesenchymal stem cells. In some other cell types (endothelium, some ureter cells) an exclusive expression of alpha 6 with no apparent colocalization of laminin A-chain in the corresponding basement membrane was seen. Thus, in these cells, integrins possessing the alpha 6 subunit may bind to laminin isoforms that differ from those synthesized by developing tubules.     

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.     

The integrin alpha 6 beta 4 is a laminin receptor

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

Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8

The involvement of integrins in mediating interaction of cells to well-characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody-sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules, possibly proteoglycans, not belonging to the integrin family.     

Isolation of alpha 6 beta 1 integrins from platelets and adherent cells by affinity chromatography on mouse laminin fragment E8 and human laminin pepsin fragment

Ligand affinity chromatography was used to identify receptors on platelets and two adherent cell lines, OV-CAR-4 and HBL-100, for the E8 fragment of murine laminin. A complex of two polypeptides (140 and 110 kDa nonreduced) was bound by the E8 affinity columns from all three cell types and was eluted with EDTA. This heterodimeric complex was identified as the alpha 6 beta 1 integrin by immunoprecipitation with specific antibodies against either the alpha 6 or the beta 1 subunit. The alpha 6 beta 1 integrin did not bind to an affinity column containing fragment P1 originating from a different part of murine laminin which, however, bound the alpha IIb beta 3 integrin from platelets. Furthermore, in immunofluorescence staining, the alpha 6 beta 1 integrin localizes in focal contacts of OVCAR-4 cells attached to laminin and E8 but not to fibronectin substrates. These results, combined with previous antibody inhibition studies, unequivocally identify the alpha 6 beta 1 integrin as a specific receptor for fragment E8. Affinity chromatography of OVCAR-4 and HBL-100 cells on a large pepsin fragment of laminin from human placenta yielded integrin alpha 3 beta 1. When alpha 3 beta 1 was removed from lysates of OVCAR-4 cells by preclearing with an alpha 3-specific monoclonal antibody, alpha 6 beta 1 was able to bind to human laminin as well. Integrin alpha 6 beta 1 on platelets which do not express alpha 3 beta 1 binds directly to human laminin. These results indicate that both alpha 3 beta 1 and alpha 6 beta 1 can act as receptors for human laminin and may interfere by steric hindrance. The alpha 6 beta 4 complex, which is strongly expressed on HBL-100 cells, did not bind to either mouse laminin fragment E8 or human laminin affinity columns.     

Expression of the integrin subunit alpha8 in murine lung development.

The complex interplay between cells and extracellular matrix (ECM) proteins is critical for lung development. Integrins are key modulators of this interaction. The integrin subunit alpha 8 associates with the beta(1)-subunit to form an RGD-binding integrin. We previously showed that, in adult lung, alpha 8 is expressed in contractile interstitial cells and smooth muscle cells and is upregulated in lung injury. To gain insight into the function of alpha 8 during lung development, we examined the spatiotemporal expression of alpha 8 throughout murine lung development. We compared the distribution of alpha 8 with alpha-smooth muscle actin (alpha SMA), fibronectin (alpha 8 ligand), and cytokeratin. alpha 8 co-localized with alpha SMA and fibronectin in the peribronchial and perivascular regions. In all stages, alpha 8 immunoreactivity was detected diffusely in the mesenchyme except for cells surrounding distal, newly forming airways. alpha 8, alpha SMA, and fibronectin co-localized at tips of secondary septae in the alveolar stage. We conclude that alpha 8 is marker for lung mesenchymal cells starting early in development. alpha 8 is also a marker for smooth muscle cells, expressed as early as alpha SMA. Co-localization of alpha 8 with fibronectin suggests a role in branching morphogenesis. Furthermore, alpha 8 may participate in secondary septation by modulating signals from the extracellular matrix to alveolar myofibroblasts.     

Regulation of laminin 1-induced pancreatic beta-cell differentiation by alpha6 integrin and alpha-dystroglycan

BACKGROUND: The ability to manipulate the development of pancreatic insulin-producing beta cells has implications for the treatment of type 1 diabetes. Previously, we found that laminin-1, a basement membrane trimeric glycoprotein, promotes beta-cell differentiation. We have investigated the mechanism of this effect, using agents that block the receptors for laminin-1, alpha6 integrin, and alpha-dystroglycan (alpha-DG). MATERIALS AND METHODS: Dissociated cells from 13.5-day postcoitum (dpc) fetal mouse pancreas were cultured for 4 days with laminin-1, with and without monoclonal antibodies and other agents known to block integrins or alpha-DG. Fetuses fixed in Bouin's solution or fetal pancreas cells fixed in 4% paraformaldehyde were processed for routine histology and for immunohistology to detect hormone expression and bromodeoxyuridine (BrdU) uptake. RESULTS: Blocking the binding of laminin-1 to alpha6 integrin with a monoclonal antibody, GoH3, abolished cell proliferation (BrdU uptake) and doubled the number of beta cells. Inhibition of molecules involved in alpha6 integrin signaling (phosphotidylinositol 3-kinase, F-actin, or mitogen-activated protein kinase) had a similar effect. Nevertheless, beta cells appeared to develop normally in alpha6 integrin-deficient fetuses. Blocking the binding of laminin-1 to alpha-DG with a monoclonal antibody, IIH6, dramatically decreased the number of beta cells. Heparin, also known to inhibit laminin-1 binding to alpha-DG, had a similar effect. In the presence of heparin, the increase in beta cells in response to blocking alpha integrin with GoH3 was abolished. CONCLUSIONS: These findings reveal an interplay between alpha6 integrin and alpha-DG to regulate laminin-1-induced beta-cell development. Laminin-I had a dominant effect via alpha-DG to promote cell survival and beta-cell differentiation, which was modestly inhibited by alpha6 signaling.     

Spermidine/spermine N1-acetyltransferase specifically binds to the integrin alpha9 subunit cytoplasmic domain and enhances cell migration

The integrin alpha9beta1 is expressed on migrating cells, such as leukocytes, and binds to multiple ligands that are present at sites of tissue injury and inflammation. alpha9beta1, like the structurally related integrin alpha4beta1, mediates accelerated cell migration, an effect that depends on the alpha9 cytoplasmic domain. alpha4beta1 enhances migration through reversible binding to the adapter protein, paxillin, but alpha9beta1-dependent migration is paxillin independent. Using yeast two-hybrid screening, we identified the polyamine catabolizing enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) as a specific binding partner of the alpha9 cytoplasmic domain. Overexpression of SSAT increased alpha9beta1-mediated migration, and small interfering RNA knockdown of SSAT inhibited this migration without affecting cell adhesion or migration that was mediated by other integrin cytoplasmic domains. The enzyme activity of SSAT is critical for this effect, because a catalytically inactive version did not enhance migration. We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates.     

Skeletal myoblasts utilize a novel beta 1-series integrin and not alpha 6 beta 1 for binding to the E8 and T8 fragments of laminin.

The E8 fragment of laminin stimulates myoblast attachment and locomotion. Myoblast attachment to laminin/E8 was blocked by anti-integrin antibodies against beta 1-chains but not by antibodies against alpha 6-chains. By contrast, other cell lines (e.g. B16, HT1080, P19, F9, Pys2, 3T3, and 3T6) were blocked both by anti-beta 1 and anti-alpha 6. All cells tested also bound to approximately 125-kDa C-terminal fragments of E8 (T8 and T8'). Immunoprecipitation of surface-iodinated myoblasts revealed beta 1-, alpha 3-, and alpha 5-integrin chains and a novel chain that co-precipitated with anti-beta 1 antibodies running at approximately 95 kDa (reduced). I125-alpha 6 beta 1 was immunoprecipitated from cells whose attachment to E8 was blocked by anti-alpha 6 antibodies. By contrast, little alpha 6 beta 1 could be immunoprecipitated from myoblasts. beta 1-Integrin and the novel alpha-chain (alpha'), Mr approximately 120,000/approximately 95.000 (nonreduced/reduced), from myoblast lysates were retained during affinity chromatography on Engelbreth-Holm-Swarm-laminin affinity columns. beta 1, alpha 1, and the novel alpha' were retained from Rugli cell lysates on Engelbreth-Holm-Swarm-laminin columns. alpha 3 was not bound. When E8 was used as affinity matrix, only beta 1 and alpha' were retained. The N-terminal sequence of Rugli alpha' was homologous to alpha-chains of beta 1-series integrins and was most similar to alpha 6 (9 identical residues out of 14). However, there were distinctive differences; in particular, 2 residues were deleted in comparison with alpha 6.     

The alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers mediate cell attachment to distinct sites on laminin

This study was undertaken to determine the roles of individual alpha/beta 1 integrin heterodimers in promoting cellular interactions with the different attachment-promoting domains of laminin (LN). To do this, antibodies to the integrin beta 1 subunit or to specific integrin alpha subunits were tested for effects on cell attachment to LN, to elastase fragments E1-4 and E1, derived from the short arms and core of LN's cruciform structure, and to fragment E8 derived from the long arm of this structure. The human JAR choriocarcinoma cells used in this study attached to LN and to fragments E1 and E8. Attachment to E1-4 required a much higher substrate coating concentration, suggesting that it is a poor substrate for JAR cell attachment. The ability of cells to attach to different LN domains suggested the presence of more than one LN receptor. These multiple LN receptors were shown to be beta 1 integrin heterodimers because antibodies to the integrin beta 1 subunit inhibited attachment of JAR cells to LN and its three fragments. To identify the individual integrin alpha/beta 1 heterodimers that mediate interactions with these LN domains, mAbs specific for individual beta 1 heterodimers in human cells were used to study JAR cell interactions with LN and its fragments. An anti-alpha 6/beta 1-specific mAb, GoH3, virtually eliminated cell attachment to E8 and partially inhibited attachment to E1 and intact LN. Thus the major alpha 6/beta 1 attachment domain is present in fragment E8. An alpha 1/beta 1-specific mAb (S2G3) strongly inhibited cell attachment to collagen IV and partially inhibited JAR attachment to LN fragment E1. Thus, the alpha 1/beta 1 heterodimer is a dual receptor for collagen IV and LN, interacting with LN at a site in fragment E1. In combination, the anti-alpha 1- and anti-alpha 6-specific antibodies completely inhibited JAR cell attachment to LN and fragment E1. Thus, the alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers each function as LN receptors and act together to mediate the interactions of human JAR choriocarcinoma cells with LN.     

Genomic organization and sequence variation of the human integrin subunit alpha8 gene (ITGA8).

The integrin alpha8 is highly expressed during kidney and lung development. alpha8-deficient mice display abnormal renal development suggesting that alpha8 plays a critical role in organogenesis. Therefore, it would be of considerable interest to understand the genomic structure, localization and sequence variation of the alpha8 gene. Using FISH and genomic database analysis, we show that alpha8 gene maps to chromosome 10p13 and consists of >200 kbp organized into 30 exons. Examination of 47 individuals from two different ethnic groups (European and African descent) identified 286 varying sites. The diversity of alpha8 is comparable to that of other regions within the human genome. Eight of the varying sites were located in the coding regions: six resulted in nonsynonymous substitutions of which two lead to non-conservative changes in protein. None of the sites showed significant deviation from Hardy-Weinberg equilibrium. We mapped the coding region single nucleotide polymorphisms (SNPs) onto a model of the predicted alpha8 structure and found all the SNPs were located in the "calf" of the extracellular domain. In the European population, the linkage disequilibrium statistic D' showed three blocks of relatively non-recombinant regions in the alpha8 gene while the African population showed more evidence of recombination. The observed patterns of the linkage disequilibrium statistic R2 suggest that a large number of sites will need to be genotyped to ensure coverage of the entire gene for genetic association studies. Identification of the sequence variation will allow genetic association studies of alpha8 in kidney and lung disease.     

The alpha4 laminin subunit regulates endothelial cell survival

The alpha4 laminin subunit is a major structural component of assembling basement membranes of endothelial cells. We have been investigating its functions with regard to endothelial cell survival. An anti-laminin alpha4 antibody (2A3), directed against the G domain of the alpha4 laminin subunit of laminins-8 and -9, inhibits proliferation and enhances apoptosis of endothelial cells when cells are maintained in vitro. Activation of caspases-9 and -3 plays a role in 2A3 antibody-induced apoptosis, since inhibitors specific for these caspases and overexpression of the anti-apoptotic protein Bcl-X(L), but not c-FLIP, inhibit 2A3 antibody-triggered endothelial cell death. Extracellular matrix is known to play a role in regulating programmed cell death in an integrin-dependent fashion. The alpha4 laminin subunit conforms to this idea since activation of beta1 integrin subunits on endothelial cells blocks the ability of 2A3 antibody to induce endothelial cell death. In summary, our data indicate that complexes composed of alpha4 laminin/beta1 subunit-containing integrins at the cell surface support endothelial cell survival. Furthermore, we propose that antagonists of alpha4 laminin function, including antibody 2A3, have value as angiogenesis inhibitors in a clinical setting where blocking aberrant growth of blood vessel by triggering apoptosis of endothelial cells may be therapeutic.     

The activation dependent adhesion of macrophages to laminin involves cytoskeletal anchoring and phosphorylation of the alpha 6 beta 1 integrin

Macrophages require activation with either PMA (Mercurio, A. M., and L. M. Shaw. 1988. J. Cell Biol. 107:1873-1880) or interferon-gamma (Shaw, L. M., and A. M. Mercurio. 1989. J. Exp. Med. 169:303-308) to adhere to a laminin substratum. In the present study, we identified an integrin laminin receptor on macrophages and characterized cellular changes that occur in response to PMA activation that facilitate laminin adhesion. A monoclonal antibody (GoH3) that recognizes the integrin alpha 6 subunit (Sonnenberg, A., H. Janssen, F. Hogervorst, J. Calafat, and J. Hilgers. 1987. J. Biol. Chem. 262:10376-10383) specifically inhibited adhesion to laminin-coated surfaces. This antibody precipitated an alpha 6 beta 1 heterodimer (Mr 130/110 kD) from 125I surface-labeled macrophages. The amount of radiolabeled receptor on the cell surface did not increase after PMA activation. Thus, the induction of laminin adhesion cannot be attributed to de novo or increased surface expression of alpha 6 beta 1. By initially removing the Triton X-100-soluble fraction of macrophages and then disrupting the remaining cytoskeletal framework, we observed that 75% of the alpha 6 beta 1 heterodimer on the cell surface is anchored to the cytoskeleton in macrophages that had adhered to a laminin substratum in response to PMA. Significant cytoskeletal anchoring of this receptor was not observed in macrophages that had adhered to fibronectin or tissue culture plastic, nor was it seen in nonadherent cells. PMA also induced phosphorylation of the cytoplasmic domain of the alpha 6 subunit, but not the beta 1 subunit. Phosphorylated alpha 6 was localized to the cytoskeletal fraction only in macrophages plated on a laminin substratum. In summary, our results support a mechanism for the regulation of macrophage adhesion to laminin that involves specific and dynamic matrix integrin-cytoskeletal interactions that may be facilitated by integrin phosphorylation.     

Characterization of mouse integrin alpha3 subunit gene.

Integrin alpha3beta1 (VLA-3) is an adhesion receptor for extracellular matrix proteins including various isoforms of laminin. We have isolated mouse genomic clones encoding the integrin alpha3 subunit and deduced the exon/intron organization. The mouse integrin alpha3 subunit gene is encoded by 26 exons spanning 40 kb. The exon/intron structure of the integrin alpha3 subunit gene resembles that of the integrin alpha6 subunit gene, but differs somewhat from those of other members of the integrin family. We have demonstrated that the cytoplasmic domain splicing variants of the alpha3 subunits (alpha3A and alpha3B) are generated by alternative exon usage. We also cloned the 5'-flanking region and performed a preliminary analysis of its promoter activity in various tumor cell lines with different degrees of integrin alpha3 expression. Following transfection, activity in the luciferase assay was found to be roughly correlated with the expression level of integrin alpha3 as measured by flow cytometry. Furthermore, the luciferase assay was performed with normal and SV-40- or polyoma virus-transformed fibroblasts. In mouse, human, and hamster fibroblasts, higher levels of luciferase expression were observed in transformed cells than in normal cells. This result is consistent with our previous finding that integrin alpha3 expression at both the protein and mRNA levels is enhanced upon oncogenic transformation of fibroblasts by tumor viruses.     

Nischarin, a novel protein that interacts with the integrin alpha5 subunit and inhibits cell migration

Integrins have been implicated in key cellular functions, including cytoskeletal organization, motility, growth, survival, and control of gene expression. The plethora of integrin alpha and beta subunits suggests that individual integrins have unique biological roles, implying specific molecular connections between integrins and intracellular signaling or regulatory pathways. Here, we have used a yeast two-hybrid screen to identify a novel protein, termed Nischarin, that binds preferentially to the cytoplasmic domain of the integrin alpha5 subunit, inhibits cell motility, and alters actin filament organization. Nischarin is primarily a cytosolic protein, but clearly associates with alpha5beta1, as demonstrated by coimmunoprecipitation. Overexpression of Nischarin markedly reduces alpha5beta1-dependent cell migration in several cell types. Rat embryo fibroblasts transfected with Nischarin constructs have "basket-like" networks of peripheral actin filaments, rather than typical stress fibers. These observations suggest that Nischarin might affect signaling to the cytoskeleton regulated by Rho-family GTPases. In support of this, Nischarin expression reverses the effect of Rac on lamellipodia formation and selectively inhibits Rac-mediated activation of the c-fos promoter. Thus, Nischarin may play a negative role in cell migration by antagonizing the actions of Rac on cytoskeletal organization and cell movement.     

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.     

A fragment of paxillin binds the alpha 4 integrin cytoplasmic domain (tail) and selectively inhibits alpha 4-mediated cell migration

The alpha(4) integrins play important roles in embryogenesis, hematopoiesis, cardiac development, and the immune responses. The alpha(4) integrin subunit is indispensable for these biological processes, possibly because the alpha(4) subunit regulates cellular functions differently from other integrin alpha subunits. We have previously reported that the alpha(4) cytoplasmic domain directly and tightly binds paxillin, an intracellular signaling adaptor molecule, and this interaction accounts for some of the unusual functional responses to alpha(4) integrin-mediated cell adhesion. We also have identified a conserved 9-amino acid region (Glu(983)-Tyr(991)) in the alpha(4) cytoplasmic domain that is sufficient for paxillin binding, and an alanine substitution at either Glu(983) or Tyr(991) within this region disrupted the alpha(4)-paxillin interaction and reversed the effects of the alpha(4) cytoplasmic domain on cell spreading and migration. In the current study, we have mapped the alpha(4)-binding site within paxillin using mutational analysis, and examined its effects on the alpha(4) tail-mediated functional responses. Here we report that sequences between residues Ala(176) and Asp(275) of paxillin are sufficient for binding to the alpha(4) tail. We found that the alpha(4) tail, paxillin, and FAT, the focal adhesion targeting domain of pp125(FAK), could form a ternary complex and that the alpha(4)-binding paxillin fragment, P(Ala(176)-Asp(275)), specifically blocked paxillin binding to the alpha(4) tail more efficiently than it blocked binding to FAT. Furthermore, when expressed in cells, this alpha(4)-binding paxillin fragment specifically inhibited the alpha(4) tail-stimulated cell migration. Thus, paxillin binding to the alpha(4) tail leads to enhanced cell migration and inhibition of the alpha(4)-paxillin interaction selectively blocks the alpha4-dependent cellular responses.     

Asn-linked oligosaccharide-dependent interaction between laminin and gp120/140. An alpha 6/beta 1 integrin

Receptor-mediated recognition and adhesion to laminin, a specific glycoprotein from basement membranes, exert an important role in many biological phenomena. Studying cell surface proteins of B16-F10, a metastatic murine melanoma cell line, we identified a 120-140 kDa glycoprotein (gp120/140) that binds laminin. This glycoprotein was recognized by a polyclonal antibody raised against the human fibronectin receptor beta 1-integrin chain, as well as immunoprecipitated by an anti-alpha 6 chain (monoclonal antibody GoH3), characterizing it as an alpha 6/beta 1-integrin. Its binding to laminin was specific and displayed moderate affinity, as its apparent dissociation constant was 18 nM. To characterize the influence of carbohydrate moieties on the laminin-gp120/140 interaction, metaperiodate oxidation, metabolic inhibition of glycosylation, and enzymatic deglycosylation studies were performed. Our results indicate that gp120/140 Asn-linked oligosaccharides play a part in this interaction. Reciprocally, both metaperiodate and N-glycanase treatment of native laminin reduced its binding to gp120/140, characterizing the latter as a lectin-like molecule. These results point to glycosylation processes as a possible mechanism for variable binding specificity profiles among integrins.     

Immunolocalization of tenascin-C, alpha9 integrin subunit, and alphavbeta6 integrin during wound healing in human oral mucosa.

Tenascin-C (TN-C) and its isoforms are multidomain extracellular matrix (ECM) proteins that are believed to be involved in the regulation of stromal-epithelial interactions. Some of the interactions between TN-C and cells are mediated by integrins. In this study we analyzed the expression of TN-C and its large molecular weight splice isoform (TN-C(L)) and the putative TN-C-binding alpha9 and alphavbeta6 integrins during human wound repair. In 3-day-old oral mucosal wounds, immunoreactivity for alpha9 integrin localized abundantly at the migrating basal wound epithelial cells. TN-C and TN-C(L) were localized in the matrix between and underneath alpha9-expressing epithelial cells. In parallel with gradual downregulation of alpha9 integrin immunoreactivity in 7-day and older wounds, the expression of alphavbeta6 integrin was temporarily induced. Integrin alphavbeta6 co-localized in the same area as TN-C and TN-C(L) immunoreactivity at the cell-cell contacts of the basal and suprabasal cell layers of the wound epithelium. During granulation tissue formation and reorganization from 7 to 28 days after wounding, TN-C and TN-C(L) were abundantly localized in the granulation tissue. The findings show that TN-C(L) is expressed under the migrating epithelial front and in the granulation tissue during matrix deposition in wound repair. Preferential localization of alpha9 integrin in migrating epithelial cells and of alphavbeta6 integrin in epithelium after wound closure suggests different functions for these integrins in wound repair.