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.     

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.     

Laminin isoforms differentially regulate adhesion, spreading, proliferation, and ERK activation of beta1 integrin-null cells

The presence of many laminin receptors of the beta1 integrin family on most cells makes it difficult to define the biological functions of other major laminin receptors such as integrin alpha6beta4 and dystroglycan. We therefore tested the binding of a beta1 integrin-null cell line GD25 to four different laminin variants. The cells were shown to produce dystroglycan, which based on affinity chromatography bound to laminin-1, -2/4, and -10/11, but not to laminin-5. The cells also expressed the integrin alpha6Abeta4A variant. GD25 beta1 integrin-null cells are known to bind poorly to laminin-1, but we demonstrate here that these cells bind avidly to laminin-2/4, -5, and -10/11. The initial binding at 20 min to each of these laminins could be inhibited by an integrin alpha6 antibody, but not by a dystroglycan antibody. Hence, integrin alpha6Abeta4A of GD25 cells was identified as a major receptor for initial GD25 cell adhesion to three out of four tested laminin isoforms. Remarkably, cell adhesion to laminin-5 failed to promote cell spreading, proliferation, and extracellular signal-regulated kinase (ERK) activation, whereas all these responses occurred in response to adhesion to laminin-2/4 or -10/11. The data establish GD25 cells as useful tools to define the role integrin alpha6Abeta4A and suggest that laminin isoforms have distinctly different capacities to promote cell adhesion and signaling via integrin alpha6Abeta4A.     

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.     

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.     

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.     

A synthetic peptide derived from the carboxy terminus of the laminin A chain represents a binding site for the alpha 3 beta 1 integrin

The purpose of this study was to identify the binding site(s) within laminin for the alpha 3 beta 1 integrin receptor. It has been previously shown, using proteolytic fragments and anti-laminin antibodies, that the region in laminin for alpha 3 beta 1 integrin binding is localized to the carboxy-terminal region at the end of the long arm (Gehlsen, K. R., E. Engvall, K. Dickerson, W. S. Argraves, and E. Ruoslahti. 1989. J. Biol. Chem. 264:19034-19038; Tomaselli, K. J., D. E. Hall, L. T. Reichardt, L. A. Flier, K. R. Gehlsen, D. C. Turner, and S. Carbonetto. 1990. Neuron. 5:651-662). Using synthetic peptides, we have identified an amino acid sequence within the carboxy-terminal region of the laminin A chain that is recognized by the alpha 3 beta 1 integrin. The amino acid sequence represented by the synthetic peptide GD-6 (KQNCLSSRASFRGCVRNLRLSR residues numbered 3011 to 3032) of the globular domain of the murine A chain supports cell attachment and inhibits cell adhesion to laminin-coated surfaces. By affinity chromatography, peptide GD-6-Sepharose specifically bound solubilized alpha 3 beta 1 from extracts of surface-iodinated cells in a cation-dependent manner, while it did not bind other integrins. In addition, exogenous peptide GD-6 specifically eluted bound alpha 3 beta 1 from laminin-Sepharose columns but did not elute the alpha 3 beta 1 integrin from a fibronectin-Sepharose column. Using integrin subunit-specific monoclonal antibodies, only those antibodies against the alpha 3 and beta 1 subunits inhibited cell adhesion to peptide GD-6-coated surfaces. Finally, a polyclonal antibody made against peptide GD-6 reacted specifically with both murine and human laminin and significantly inhibited cell adhesion to laminin-coated surfaces but not those coated with other matrix proteins. These results identify the laminin A chain amino acid sequence of peptide GD-6 as representing a binding site in laminin for the alpha 3 beta 1 integrin.     

Identification of the alpha6 integrin as a candidate receptor for papillomaviruses.

Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction of human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [35S]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The alpha6beta4 integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the alpha6 or beta4 integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An alpha6 integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-beta4 integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the alpha6beta4 integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of alpha6 integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the alpha6 integrin subunit and that integrin complexes containing alpha6 integrin complexed with either beta1 or beta4 integrins may act as a receptor for PV binding and entry into epithelial cells.     

Interaction of the disintegrin and cysteine-rich domains of ADAM12 with integrin alpha7beta1

We describe a novel interaction between the disintegrin and cysteine-rich (DC) domains of ADAM12 and the integrin alpha7beta1. Integrin alpha7beta1 extracted from human embryonic kidney 293 cells transfected with alpha7 cDNA was retained on an affinity column containing immobilized DC domain of ADAM12. 293 cells stably transfected with alpha7 cDNA adhered to DC-coated wells, and this adhesion was partially inhibited by 6A11 integrin alpha7 function-blocking antibody. The X1 and the X2 extracellular splice variants of integrin alpha7 supported equally well adhesion to the DC protein. Integrin alpha7beta1-mediated cell adhesion to DC had different requirements for Mn2+ than adhesion to laminin. Furthermore, integrin alpha7beta1-mediated cell adhesion to laminin, but not to DC, resulted in efficient cell spreading and phosphorylation of focal adhesion kinase (FAK) at Tyr397. We also show that adhesion of L6 myoblasts to DC is mediated in part by the endogenous integrin alpha7beta1 expressed in these cells. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulate that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle development, function, and disease. We also conclude that laminin and the DC domain of ADAM12 represent two functional ligands for integrin alpha7beta1, and adhesion to each of these two ligands via integrin alpha7beta1 triggers different cellular responses.     

Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium.     

Distinct functions for integrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes

Basal cells of stratified epidermis are anchored to the basement membrane zone (BMZ) of skin via hemidesmosomes. We previously identified integrin alpha 3 beta 1, in focal adhesions (FAs), of cultured human keratinocytes (HFKs) as a mediator of HFK adhesion to secreted BMZ-like extracellular matrix (ECM; Carter, W.G., E.A. Wayner, T.S. Bouchard, and P. Kaur. 1990. J. Cell Biol. 110: 1387-1404). Here, we have examined the relation of integrins alpha 6 beta 4 and alpha 3 beta 1, to bullous pemphigoid antigen (BPA), a component of hemidesmosomes. We conclude that alpha 6 beta 4 in HFKs localizes in a new stable anchoring contact (SAC) that cooperates with alpha 3 beta 1- FAs to mediate adhesion to ECM, based on the following. (a) Comparison of secreted ECM, with exogenous laminin, fibronectin and collagen identified ECM as the preferred ligand for HFK adhesion and spreading and for formation of both alpha 6 beta 4-SACs and alpha 3 beta 1-FAs. (b) Inhibition of HFK adhesion with combined anti-alpha 3 beta 1 (P1B5) and anti-alpha 6 beta 4 (GoH3) antibodies indicated that both receptors were functional in adhesion to ECM while alpha 3 beta 1 played a dominant role in spreading. (c) alpha 6 beta 4 colocalized with BPA in SACs that were proximal to but excluded from FAs. Both alpha 6 beta 4- SACs and alpha 3 beta 1-FAs were in contact with the adhesion surface as indicated by antibody exclusion and interference reflection microscopy. (d) In contrast to alpha 3 beta 1-FAs, alpha 6 beta 4-SACs were present only in nonmotile cells, not associated with stress fibers, and were relatively stable to detergents and urea, suggesting a nonmotile, or anchoring function for SACs and motility functions for alpha 3 beta 1-FAs. (e) alpha 6 beta 4 formed a detergent-insoluble complex with exogenous ECM in an affinity isolation procedure, confirming the ability of an unidentified ECM ligand to interact with alpha 6 beta 4. (f) We suggest that alpha 6 beta 4/BPA-SACs in culture restrict migration of HFKs on ECM while alpha 3 beta 1-FAs form dynamic adhesions in spreading and migrating cells. alpha 6 beta 4/BPA-SACs in culture bear functional and compositional similarities to hemidesmosomes in skin.     

Multiple integrins share the ability to induce elevation of intracellular pH

Previous work has shown that adhesion of anchorage-dependent cells to fibronectin via integrin alpha 5 beta 1 leads to activation of the Na-H antiporter and a rise in intracellular pH (pHi). We now show that adhesion of bovine capillary endothelial cells (BCE) to fibrinogen; collagens type III, IV, and V; laminin; and vitronectin; ligands that bind other members of the integrin family, resulted in significant elevations in pHi. Other ligands (basic fibroblast growth factor, concanavalin A, and thrombin), which bind cells when immobilized on plastic, but that do not bind integrins and do not support cell growth, do not elevate pHi. Adhesion to an antibody against integrin alpha v beta 3 also elevates pHi. Adhesion of peripheral human T lymphocytes to an antibody against the integrin LFA-1 induced a rise in pHi. Antibodies to CD2 or ICAM-2 had only slight effects on pHi, whereas an antibody to the T cell receptor complex that strongly activates T cells induced a large increase in pHi. We conclude that elevation of pHi by integrins is specific and is a property shared by many members of the integrin family.     

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.     

Keratinocyte apoptosis on type I collagen gel caused by lack of laminin 5/10/11 deposition and Akt signaling

Cultured human foreskin keratinocytes (HFKs) adhere to and grow on nonfibrous collagen via integrin alpha2beta1. During incubation, the receptors used for adhesion are changed to integrins alpha3beta1 and alpha6beta4 and those receptors bind to laminin 5 which is deposited by keratinocytes themselves. In this report, we examined the behaviors of HFKs and transformed keratinocytes on collagen fibril gels. These cells adhered to and spread on collagen gels using integrin alpha2beta1. After several hours on collagen gels, however, cells became round and apoptosis occurred. The behavior of keratinocytes contrasted to that of fibroblasts that grew well even on collagen gel. At the point of apoptosis, integrins alpha2beta1 and alpha3beta1 were not found in the contact region of HFKs. Also, deposition of laminin 5 on collagen gel was not found despite the synthesis of mRNA for laminin 5 and laminin 10/11, while soluble laminin 5 protein is readily detectable. Phosporylation of Akt, which is known as a survival signal, was detected in HFKs cultured on coated collagen; however, the protein level and signals of Akt were dramatically decreased on collagen gel after 1 day of culture. These results indicate that collagen gel has different effects than nonfibrous collagen on HFKs and transformed keratinocytes and the interactions of integrin alpha3beta1 and laminin 5/10/11 are indispensable for maintenance of keratinocyte adhesion and survival.     

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.     

Analysis of the roles of RGD-binding integrins, alpha(4)/alpha(9) integrins, alpha(6) integrins, and CD9 in the interaction of the fertilin beta (ADAM2) disintegrin domain with the mouse egg membrane

Fertilin beta (also known as ADAM2), a mammalian sperm protein that mediates gamete cell adhesion during fertilization, is a member of the ADAM protein family whose members have disintegrin domains with homology to integrin ligands found in snake venoms. Fertilin beta utilizes an ECD sequence within its disintegrin domain to interact with the egg plasma membrane; the Asp is especially critical. Based on what is known about different integrin subfamilies and their ligands, we sought to characterize fertilin beta binding sites on mouse eggs, focusing on integrin subfamilies that recognize short peptide sequences that include an Asp residue: the alpha(5)/alpha(8)/alpha(v)/alpha(IIb) or RGD-binding subfamily (alpha(5)beta(1), alpha(8)beta(1), alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(5), alpha(V)beta(6), alpha(V)beta(8), and alpha(IIb)beta(3)) and the alpha(4)/alpha(9) subfamily (alpha(4)beta(1), alpha(9)beta(1), and alpha(4)beta(7)). We tested peptide sequences known to perturb interactions mediated by these integrins in two different assays for fertilin beta binding. Peptides with the sequence MLDG, which perturb alpha(4)/alpha(9) integrin-mediated interactions, significantly inhibit fertilin beta binding to eggs, which suggests a role for a member of this integrin subfamily as a fertilin beta receptor. RGD peptides, which perturb alpha(5)/alpha(8)/alpha(v)/alpha(IIb) integrin-mediated interactions, have partial inhibitory activity. The anti-alpha(6) antibody GoH3 has little or no inhibitory activity. An antibody to the integrin-associated tetraspanin protein CD9 inhibits the binding of a multivalent presentation of fertilin beta (immobilized on beads) but not soluble fertilin beta, which we speculate has implications for the role of CD9 in the strengthening of fertilin beta-mediated cell adhesion but not in initial ligand binding.     

A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins

To examine the function of the alpha 6 beta 4 integrin we have determined its ligand-binding ability and overexpressed two potentially dominant negative mutant beta 4 subunits, lacking either the cytoplasmic or extracellular domain, in bladder epithelial 804G cells. The results of cell adhesion and radioligand-binding assays showed that alpha 6 beta 4 is a receptor for several laminin isoforms, including laminin 1, 2, 4, and 5. Overexpression of the tail-less or head-less mutant beta 4 subunit did not suppress alpha 6 beta 4-mediated adhesion to laminins, as both types of transfectants adhered to these ligands in the presence of blocking anti-beta 1 antibodies as well as the controls. However, immunofluorescence experiments indicated that the endogenous alpha 6 beta 4 integrin and other hemidesmosomal markers were not concentrated in hemidesmosomes in cells overexpressing tail- less beta 4, while the distribution of these molecules was not altered in cells overexpressing the head-less subunit. Electron microscopic studies confirmed that cells overexpressing tail-less beta 4 had a drastically reduced number of hemidesmosomes, while cells expressing the head-less subunit had a normal number of these structures. Thus, expression of a tail-less, but not a head-less mutant beta 4 subunit leads to a dominant negative effect on hemidesmosome assembly without suppressing initial adhesion to laminins. We conclude that the alpha 6 beta 4 integrin binds to several laminins and plays an essential role in the assembly and/or stability of hemidesmosomes, that alpha 6 beta 4- mediated adhesion and hemidesmosome assembly have distinct requirements, and that it is possible to use a dominant negative approach to selectively interfere with a specific function of an integrin.     

Alpha 1 beta 1 integrin heterodimer functions as a dual laminin/collagen receptor in neural cells

A monoclonal antibody (3A3) raised against a rat neural cell line (PC12) was shown previously to bind to the surfaces of these cells, inhibiting substratum adhesion. Immunochemical and other data indicated that the heterodimer recognized by 3A3 was a member of the integrin family of adhesive receptors and had a beta 1 subunit. The relationship of the alpha subunit to other integrins was unknown. Here we show that 3A3 recognizes in rat tissues a heterodimer (approximately 185 kDa, approximately 110 kDa; unreduced) that is electrophoretically and immunochemically indistinguishable from the antigen in PC12 cells. Immunoaffinity purification of the heterodimer from neonatal rats and protein microsequencing indicate that the alpha subunit is identical at 11 or 13 N-terminal residues with VLA-1, an integrin on human hematopoietic cells. Monoclonal antibody 3A3 inhibits the attachment of rat astrocytes to laminin or collagen but not to fibronectin or polylysine. These data suggest strongly that the integrin recognized by 3A3 is the rat homologue of VLA-1, i.e., alpha 1 beta 1, and that alpha 1 beta 1 is a dual laminin/collagen receptor.     

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.