myospheroid

EVOLUTIONARY HOMOLOGS

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Other integrin-ligand interactions

Nine integrin alpha subunits contain an additional domain (termed A or I) that is inserted into the head region, where it plays a central role in ligand binding: thus, recombinant I domains recapitulate many of the ligand-binding properties of the intact integrin. The first crystal structure of an I domain shows that it adopts the 'dinucleotide-binding' fold, with a central mostly parallel beta sheet surrounded on both sides by amphipathic alpha helices. At the C-terminal end of the beta sheet is a conserved metal binding site that has been called the metal ion-dependent adhesion site, or MIDAS motif. Mutagenesis studies have shown that the MIDAS motif and exposed side chains on the surrounding surface are required for ligand binding, and are thus likely to form the ligand contact sites. Comparison between two different crystal forms of the alphaM-I domain led to the proposal that affinity regulation occurs via changes in metal coordination at the MIDAS motif that are linked to tertiary changes in the domain. The crystal structure of a complex between the I domain of integrin alpha2beta1 and a triple helical collagen peptide containing a critical GFOGER motif has been determined. Three loops on the upper surface of the I domain that coordinate a metal ion also engage the collagen, with a collagen glutamate completing the coordination sphere of the metal. Comparison with the unliganded I domain reveals a change in metal coordination linked to a reorganization of the upper surface that together create a complementary surface for binding collagen. Conformational changes propagate from the upper surface to the opposite pole of the domain, suggesting both a basis for affinity regulation and a pathway for signal transduction. The structural features observed here may represent a general mechanism for integrin-ligand recognition (Emsley, 2000).

alpha11beta1 integrin constitutes a recent addition to the integrin family. The first in vivo analysis of alpha11 protein and mRNA distribution during human embryonic development is presented. alpha11 protein and mRNA are present in various mesenchymal cells around the cartilage anlage in the developing skeleton in a pattern similar to that described for the transcription factor scleraxis. alpha11 is also expressed by mesenchymal cells in intervertebral discs and in keratocytes in cornea, two sites with highly organized collagen networks. Neither alpha11 mRNA nor alpha11 protein could be detected in myogenic cells in human embryos. The described expression pattern is compatible with alpha11beta1 functioning as a receptor for interstitial collagens in vivo. To test this hypothesis in vitro, full-length human alpha11 cDNA was stably transfected into the mouse satellite cell line C2C12, which lacks endogenous collagen receptors. alpha11beta1 mediated cell adhesion to collagens I and IV (with a preference for collagen I) and formed focal contacts on collagens. In addition, alpha11beta1 mediated contraction of fibrillar collagen gels in a manner similar to alpha2beta1, and supported migration on collagen I in response to chemotactic stimuli. These data support a role for alpha11beta1 as a receptor for interstitial collagens on mesenchymally derived cells and suggest a multifunctional role of alpha11beta1 in the recognition and organization of interstitial collagen matrices during development (Tiger, 2001).

The astonishing similarity between the structural changes seen in the alpha2- and alphaM-I domains suggests that there are just two principal conformations for I domains: 'open' and 'closed'. The open conformation is seen in the presence of ligand or ligand mimetic, while the closed conformation is seen in the absence of ligand. What triggers the conformational switch appears to be the formation of a strong ligand-metal bond, requiring a change in metal coordination. While lack of surface complementarity is likely to play a role, it is suggested that a major barrier to high-affinity ligand binding in the closed conformation is the direct coordination of the metal by an aspartic acid from the I domain, which reduces its electrophilicity. In the case of the alpha2-I domain, the C helix provides an additional steric barrier in the closed conformation to the attainment of the metal-ligand bond. Other studies support the hypothesis that the open and closed conformations equate with high- and low-affinity states. Mutants of the alphaM-I domain that are predicted on structural grounds to destabilize the closed conformation and favor the open conformation increase the affinity for the ligand C3bi. The epitope for an antibody that binds only to the high-affinity form of the alphaMbeta2 integrin maps to loop 1 and helix alpha7 of the I domain, a region that undergoes extensive conformational changes between the closed and open forms. These observations suggest that the conformational changes observed in crystals of the recombinant I domain also occur within the context of the parent molecule. This in turn suggests that affinity regulation in the integrin is provided by quaternary contacts that restrain these conformational changes in the I domain, and, conversely, that ligand binding could provide the trigger for quaternary structural changes (Emsley, 2000 and references therein). *

Several lines of evidence point to the quaternary contacts occurring between the body of the integrin and the lower surface of the I domain, at the opposite pole from the ligand-binding surface. Thus, mutations that lead to a constitutively active integrin map to this lower surface, presumably by destabilizing the interface that normally locks the C-terminal helix in the closed conformation. The recent structure determination of the alphaL-I domain in complex with the inhibitor lovastatin reveals that lovastatin binds in a hydrophobic pocket at the base of the domain, between the beta sheet and the C-terminal helix: it is suggested that lovastatin inhibits alphaLbeta2 integrin function allosterically by preventing the C-terminal helix shift required for the domain to adopt the open conformation that would bind ICAM with high affinity. These data are highly reminiscent of the natural type IIB mutants in the plasma protein von Willebrand Factor that lead to constitutively high-affinity ligand binding. The A1 domain of vWF has an identical fold to the integrin I domain (although it does not bind metal), with the ligand binding surface at the top of the domain and the activating mutations at the bottom. A common feature here is that the A/I domains function in the plasma and are involved in the tightly regulated binding of cells to the subendothelial matrix under conditions of high flow/shear. The high-affinity conformation, with its large movement of the C-terminal helix, is more extended or 'stretched' than the low-affinity conformer. The existence of bonds that strengthen when tensile force is applied ('catch bonds') have been predicted theoretically. The conformational stretching observed here may suggest a molecular basis for such bonds (Emsley, 2000 and references therein).

These results suggest that the metal bridge between the integrin and an acidic residue in the ligand will be a general and critical feature of I domain-ligand interactions. Not all integrins possess an alpha subunit I domain, but they all contain a region in their beta subunits that is proposed to contain a MIDAS-like motif. In the beta3 integrins, for example, this is the site for binding the matrix proteins containing the RGD motif. It seems increasingly likely that the aspartic acid of the RGD motif will coordinate a metal ion in a similar fashion to that observed here for the collagen glutamate, and that analogous conformational changes triggered by a subtle change in metal coordination will underlie affinity regulation and signal transduction in all integrins (Emsley, 2000 and references therein).

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38-61 and 82-101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-alphavbeta3 antibodies prevent cell adhesion to FGF-2. Purified human alphavbeta3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Anti-alphavbeta3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with alphavbeta3 integrin. This interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells (Rusnati, 1997).

Regulation of vascular homeostasis depends on collaboration between cells of the vessel wall and blood coagulation system. A direct interaction between integrin alphaVbeta3 on endothelial cells and smooth muscle cells and prothrombin, the pivotal proenzyme of the blood coagulation system, has been demonstrated; activation of the integrin is required for receptor engagement. Evidence that prothrombin is a ligand for alphaVbeta3 on these cells includes: (1) prothrombin binds to purified alphaVbeta3 via a RGD recognition specificity; (2) prothrombin supports alphaVbeta3-mediated adhesion of stimulated endothelial cells and smooth muscle cells, and (3) endothelial cells, either in suspension and in a monolayer, recognize soluble prothrombin via alphaVbeta3. alphaVbeta3-mediated cell adhesion to prothrombin, but not to fibrinogen, requires activation of the receptor. Thus, the functionality of the alphaVbeta3 receptor is ligand defined, and prothrombin and fibrinogen represent activation-dependent and activation-independent ligands. Activation of alphaVbeta3 can be induced not only by model agonists, PMA and Mn2+, but also by a physiologically relevant agonist, ADP. Inhibition of protein kinase C and calpain prevents activation of alphaVbeta3 on vascular cells, suggesting that these molecules are involved in the inside-out signaling events that activate the integrin. The capacity of alphaVbeta3 to interact with prothrombin may play a significant role in the maintenance of hemostasis; and, at a general level, ligand selection by alphaVbeta3 may be controlled by the activation state of this integrin (Byzova, 1998).

The multipotential cytokine transforming growth factor-beta (TGF-beta) is secreted in a latent form. Latency results from the noncovalent association of TGF-beta with its processed propeptide dimer, called the latency-associated peptide (LAP); the complex of the two proteins is termed the small latent complex. Disulfide bonding between LAP and latent TGF-beta-binding protein (LTBP) produces the most common form of latent TGF-beta, the large latent complex. The extracellular matrix (ECM) modulates the activity of TGF-beta. LTBP and the LAP propeptides of TGF-beta (isoforms 1 and 3), like many ECM proteins, contain the common integrin-binding sequence RGD. Determining whether or not latent TGF-beta1 interacts with integrins would further an understanding of latent TGF-beta function in the ECM. A549 cells adhere and spread on plastic coated with LAP, small latent complex, and large latent complex but not on LTBP-coated plastic. Adhesion is blocked by an RGD peptide, and cells are unable to attach to a mutant form of recombinant LAP lacking the RGD sequence. Adhesion is also blocked by mAbs to integrin subunits alphav and beta1. LAP-binding integrins were purified from extracts of A549 cells using LAP bound to Sepharose. alphavbeta1 elutes with EDTA. After purification in the presence of Mn2+, a small amount of alphavbeta5 is also detected. A549 cells migrate equally on fibronectin- and LAP-coated surfaces; migration on LAP is alphavbeta1 dependent. These results establish alphavbeta1 as a LAP-beta1 receptor. Interactions between latent TGF-beta and alphavbeta1 may localize latent TGF-beta to the surface of specific cells and may allow the TGF-beta1 gene product to initiate signals by both TGF-beta receptor and integrin pathways (Munger, 1998).

Growth factors modulate integrin-mediated cell adhesion and motility; their receptors are thought to share proteins that mediate intracellular signaling with integrin receptors. The crosstalk between these receptors is thought to play a relevant role in transformation and tumor progression. To highlight possible interactions between growth factors and cell adhesion receptors, an investigation was carried out to determine if integrins associate with tyrosine kinase receptors in tumor cells. The association of laminin receptors (alpha 6 beta 1 and alpha 6 beta 4) with ErbB-2 tyrosine kinase was examined in human carcinoma cell lines. The alpha 6 beta 4 and alpha 6 beta 1 integrins coprecipitate with ErbB-2 in lysates from carcinoma or NIH3T3 cells overexpressing ErbB-2. Integrin-mediated activation of ErbB-2 receptors suggest that this association is functionally meaningful. Indeed, carcinoma cells treated with a monoclonal antibody to the alpha 6 integrin subunit show a ligand-dependent increase of ErbB-2-phosphorylated molecules coprecipitated with integrins and an increased DNA synthesis. The interaction between growth factor receptors and integrins was also studied in NIH3T3 cells overexpressing alpha 6 beta 4 receptors and ErbB-2 protein. Cells overexpressing both receptors, but not those overexpressing a crippled ErbB-2, show enhanced proliferation rates and invasiveness, further suggesting that alpha 6 beta 4 integrin and ErbB-2 receptor interaction might contribute to generate a more malignant phenotype in carcinoma cells (Falcioni, 1997).

Two distinct populations of myoblasts, distinguishable by alpha7 integrin expression have been hypothesized to give rise to two phases of myofiber formation in embryonic limb development. Alpha7 integrin is detectable far earlier than previously reported on both 'primary' and 'secondary' lineage myoblasts and myofibers. An antibody (1211) that recognizes an intracellular epitope allows detection of alpha7 integrin previously missed, using an antibody (H36) that recognizes an extracellular epitope. When myoblasts were isolated and cultured from different developmental stages, H36 only detects alpha7 integrin that is in direct contact with its ligand, laminin. Moreover, alpha7 integrin detection by H36 is reversible and highly localized to subcellular points of contact between myoblasts and laminin-coated 2.8-mm microspheres. Prior to secondary myofiber formation in limb embryogenesis, laminin is present but not in close proximity to clusters of primary myofibers that expressed alpha7 integrin detected by antibody 1211, using deconvolution microscopy. These results suggest that the timing of the interaction of preexisting alpha7 integrin with its ligand, laminin, is a major determinant of allosteric changes that result in an activated form of alpha7 integrin capable of transducing signals from the extracellular matrix commensurate with secondary myofiber formation (Blanco-Bose, 2001).

Laminin 5 (alpha3beta3gamma2) distribution in the human thymus was investigated by immunofluorescence on frozen sections with anti-alpha3, -beta3, and -gamma2 mAbs. In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures. Laminin 5 may influence mature human thymocyte expansion. Although bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect on mitosis. By contrast, soluble laminin 5 inhibits thymocyte proliferation induced by a TCR signal. This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck). Using a mAb specific for alpha6beta4 integrins, it was observed that while alpha3beta1 is known to be uniformly present on all thymocytes, alpha6beta4 expression parallels thymocyte maturation; thus a correspondence exists between laminin 5 in the thymic medulla and alpha6beta4 on mature thymocytes. Moreover, the soluble Ab against alpha6beta4 inhibits thymocyte proliferation and reproduces the same pattern of tyrosine kinase phosphorylation suggesting that alpha6beta4 is involved in laminin 5-induced modulation of T cell activation (Vivinus-Nebot, 1999).

Wounding of skin activates epidermal cell migration over exposed dermal collagen and fibronectin and over laminin 5 secreted into the provisional basement membrane. Gap junctional intercellular communication (GJIC) has been proposed to integrate the individual motile cells into a synchronized colony. Outgrowths of human keratinocytes in wounds or epibole cultures display parallel changes in the expression of laminin 5, integrin alpha3beta1, E-cadherin, and the gap junctional protein connexin 43. Adhesion of keratinocytes on laminin 5, collagen, and fibronectin differentially regulates GJIC. When keratinocytes are adhered on laminin 5, both structural (assembly of connexin 43 in gap junctions) and functional (dye transfer) assays show a two- to three-fold increase compared with collagen and five- to eight-fold over fibronectin. Based on studies with immobilized integrin antibody and integrin-transfected Chinese hamster ovary cells, the interaction of integrin alpha3beta1 with laminin 5 is sufficient to promote GJIC. Mapping of intermediate steps in the pathway linking alpha3beta1-laminin 5 interactions to GJIC indicate that protein trafficking and Rho signaling are both required. It is suggested that adhesion of epithelial cells to laminin 5 in the basement membrane via alpha3beta1 promotes GJIC, which integrates individual cells into synchronized epiboles (Lampe, 1998).

An enhancer trap event in a line of transgenic mice was used to identify a unique developmentally regulated endothelial cell locus (Del1). The protein encoded in this locus contains three EGF-like repeats homologous to those in Notch and related proteins, including an EGF-like repeat that contains an RGD motif, and two discoidin I-like domains. Del1 is shown to be a matrix protein that promotes adhesion of endothelial cells through interaction with the alphavbeta3 integrin receptor. Embryonic endothelial-like yolk sac cells expressing recombinant Del1 protein, or grown on an extracellular matrix containing Del1 protein, are inhibited from forming vascular-like structures. Expression of Del1 protein in the chick chorioallantoic membrane leads to loss of vascular integrity and promotes vessel remodeling. Del1 is thus a new ligand for the alphavbeta3 integrin receptor and may function to regulate vascular morphogenesis or remodeling in embryonic development (Hidai, 1998).

The extracellular matrix (ECM) activates signaling pathways that control cell behavior by binding to cell-surface integrin receptors and inducing the formation of focal adhesion complexes (FACs). In addition to clustered integrins, FACs contain proteins that mechanically couple the integrins to the cytoskeleton and to immobilized signal-transducing molecules. Cell adhesion to the ECM also induces a rapid increase in the translation of preexisting messenger RNAs. Gene expression can be controlled locally by targeting mRNAs to specialized cytoskeletal domains. This study sought to determine whether cell binding to the ECM promotes formation of a cytoskeletal microcompartment specialized for translational control at the site of integrin binding. High-resolution in situ hybridization showed that mRNA and ribosomes rapidly and specifically localized to FACs that form when cells bind to ECM-coated microbeads. Relocation of these protein synthesis components to the FAC depends on the ability of integrins to mechanically couple the ECM to the contractile cytoskeleton and on associated tension-moulding of the actin lattice. These results suggest a new type of gene regulation by integrins and by mechanical stress that may involve translation of mRNAs into proteins near the sites of signal reception (Chicurel, 1998).

Development of the metanephric kidney crucially depends on proper interactions between cells and the surrounding extracellular matrix. For example, in the absence of α8ß1 integrin, invasion by the ureteric bud into the metanephric mesenchyme is inhibited, resulting in renal agenesis. Genetic evidence is presented that the extracellular matrix protein nephronectin is an essential ligand that engages α8ß1 integrin during early kidney development. Embryos lacking a functional nephronectin gene frequently display kidney agenesis or hypoplasia, which can be traced to a delay in the invasion of the metanephric mesenchyme by the ureteric bud at an early stage of kidney development. Significantly, no defects in extracellular matrix organization were detected in the nascent kidneys of the nephronectin mutants. Instead, Gdnf expression was dramatically reduced in both nephronectin- and α8 integrin-null mutants specifically in the metanephric mesenchyme at the time of ureteric bud invasion. This reduction is sufficient to explain the agenesis and hypoplasia observed in both mutants. Interestingly, the reduction in Gdnf expression is transient, and its resumption presumably enables the nephronectin-deficient ureteric buds to invade the metanephric mesenchyme and begin branching. Thus, these results place nephronectin and α8ß1 integrin in a pathway that regulates Gdnf expression and is essential for kidney development (Linton, 2007).

Recognition of Netrin-1 by integrins regulates epithelial cell adhesion and migration

Netrins, axon guidance cues in the CNS, have also been detected in epithelial tissues. In this study, using the embryonic pancreas as a model system, Netrin-1 is shown to be expressed in a discrete population of epithelial cells, localizes to basal membranes, and specifically associates with elements of the extracellular matrix. α6β4 integrin mediates pancreatic epithelial cell adhesion to Netrin-1, whereas recruitment of α6β4 and α3β1 regulate the migration of CK19⁺/PDX1⁺ putative pancreatic progenitors on Netrin-1. These results provide evidence for the activation of epithelial cell adhesion and migration by a neural chemoattractant, and identify Netrin-1/integrin interactions as adhesive/guidance cues for epithelial cells (Yebra, 2003).

Integrins α6β4 and α3β1 are laminin receptors primarily expressed in epithelial cells that have been implicated in branching morphogenesis, development, and function of epithelial tissues. In addition to promoting cell adhesion, α6β4 has been involved in signaling that regulates cell survival, proliferation, and migration. Interestingly, α3β1 can cooperate with α6β4 to mediate cell motility, following activation by growth factors such as HGF/SF. The results show that α6β4 plays a major role in epithelial cell adhesion to Netrin-1. This interaction can be mapped to a highly basic region of the Netrin-1 C terminus similar to basic residue-rich domains of laminins, known to support recognition and binding of integrins α3β1, α6β1, and α6β4. The observation that classical Netrin-1 receptors DCC and Neogenin do not mediate adhesion to Netrin-1 may be explained by the extremely low levels of expression of these two receptors detected in the pancreatic epithelium, and supports a primary role of integrins α3β1 and α6β4 in the recognition of Netrin-1 (Yebra, 2003).

Integrins and Hemidesmosomes

The integrin alpha6 beta4 is a major component of hemidesmosomes; as a part of this structure, integrin alpha6 beta4 mediates firm adhesion to laminin 5. Previous studies have shown that the incorporation of alpha6 beta4 into hemidesmosomes requires a 303 amino acid stretch of the cytoplasmic domain of beta4, comprising part of the first fibronectin type III (FNIII) repeat, the second FNIII repeat and the segment that connects the second to the third FNIII repeat (connecting segment). The sequences within beta4 that are critical for its localization in hemidesmosomes have been further defined. These sequences also induce the redistribution of HD1/plectin into junctional complexes containing the integrin alpha6 beta4 in COS-7 cells, transfected with cDNAs encoding alpha6A and beta4. Truncation of the cytoplasmic domain of beta4 after amino acids 1,382 or 1,355 in the connecting segment, by which a potential tyrosine activation motif (TAM) is removed, does not prevent the localization of alpha6 beta4 in hemidesmosomes in the rat bladder carcinoma cell line 804G; neither does it eliminate the ability of alpha6 beta4 to change the subcellular distribution of HD1/plectin in COS-7 cells. In contrast, beta4 subunits in which the entire connecting segment has been deleted or which are truncated after amino acid 1,328, which removes almost the complete segment, have lost both of these functions. When beta4 subunits with either a deletion of the second FNIII repeat or a small deletion in this repeat are co-expressed with alpha6, the integrins are not localized in hemidesmosomes and do not induce the redistribution of HD1/plectin in COS-7 cells. The fourth FNIII repeat of beta4 cannot replace the second in either of these activities. These findings establish that a region in beta4, which encompasses the second FNIII repeat and a stretch of 27 amino acids (1,329-1,355) of the connecting segment, are critical for the localization of alpha6beta4 in hemidesmosomes; these regions regulate the distribution of HD1/plectin (Niessen, 1997).

Disintegrins

Kuzbanian, a Drosophila neurogenic gene (Frambrough, 1996), is a metalloprotease-disintegrin with highly conserved mammalian homologs. ADAM (standing for A Disintegrin And Metalloprotease) domains are unique among cell surface proteins in possessing both a potential adhesion domain and a protease domain. The name ADAM honors the dual origins of research that have added to the scientific tree of knowledge concerning these proteins: the fields of fertility and snakes. The first ADAMs described, fertilin alpha and beta, are expressed in spermatogonic cells. One ADAM has been implicated in integrin-mediated sperm-egg binding. Domains in ADAMs are also related to domains found in a family of soluble snake venom proteins, the snake venom metalloproteases. Snake venom metalloproteases and disintegrins promote hemorrhage in snake bite victims. Soluble metalloproteases degrade capillary basement membranes, and soluble disintegrins bind to platelet integrins, thereby inhibiting platelet aggregation (Wolfsberg, 1995).

Integrins can exist in different functional states with low or high binding capacity for particular ligands. The integrin alpha6beta1, on mouse eggs and on alpha6-transfected cells, interacts with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin beta). The hypothesis was tested that different states of alpha6beta1 interact with fertilin and laminin, an extracellular matrix ligand for alpha6beta1. Using alpha6-transfected cells it was found that treatments (e.g., with phorbol myristate acetate or MnCl2) that increase adhesion to laminin inhibit sperm binding. Conversely, treatments that inhibit laminin adhesion increase sperm binding. The ability of fluorescent beads coated with either fertilin beta or with the laminin E8 fragment to bind to eggs was examined. In Ca2+-containing media, fertilin beta beads bind to eggs via an interaction mediated by the disintegrin loop of fertilin beta and by the alpha6 integrin subunit. In Ca2+-containing media, laminin E8 beads do not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibits fertilin bead binding, but does not induce laminin E8 bead binding. Treatment of eggs with Mn2+ dramatically increases laminin E8 bead binding, and inhibits fertilin bead binding. These results provide the first evidence that different states of an integrin (alpha6beta1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2) (Chen, 1999).

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