Localization of alpha 7 integrins and dystrophin suggests potential for both lateral and longitudinal transmission of tension in large mammalian muscles

Non-primate mammalian muscles with fascicles above 35 mm in length are composed predominantly of arrays of short, non-spanning muscle fibres, which terminate within the belly of the muscle fascicle at one or both ends. We have previously described the morphological form of various muscle-to-muscle and muscle-to-matrix junctions which are likely involved in tension transmission within one such muscle - the guinea pig sternomastoid muscle (Young et al. 2000). Here, we use immunohistochemistry to investigate the cell adhesion molecules present at these junctions. We find strong immunoreactivity against the alpha 7B integrin subunit and dystrophin, and slight reactivity against the alpha 7A integrin at all intrafascicular fibre terminations (IFTs), as well as at the muscle-tendon junction (MTJ). Tenascin, the sole ligand for alpha 9 beta 1 integrin, was absent from IFTs but present at the MTJ, suggesting the two sites are molecularly distinct. In addition to their expression at junctional sites, alpha 7B integrin and dystrophin were also expressed ubiquitously along the non-junctional sarcolemma, suggesting potential involvement in diffuse lateral transmission of tension between adjacent fibres. We conclude that the distribution of alpha 7 beta 1 integrins and dystrophin in series-fibred muscles suggests they are involved in transmission of tension from intrafascicularly terminating fibres to neighbouring fibres lying both in-series and in-parallel, via the extracellular matrix (ECM).     

Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice

Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the alpha 7 beta 1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD), alpha 2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and alpha 7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the alpha 7 beta 1 integrin can compensate for the absence of dystrophin, we expressed the rat alpha 7 chain in mdx/utr(-/-) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the alpha 7BX2 integrin chain was increased 2.0-2.3-fold in mdx/utr(-/-) mice. Concomitant with the increase in the alpha 7 chain, its heterodimeric partner, beta 1D, was also increased in the transgenic animals. Transgenic expression of the alpha 7BX2 chain in the mdx/utr(-/-) mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering alpha 7 beta 1 integrin-mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr(-/-) mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the alpha 7 beta 1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr(-/-) and alpha 7BX2-mdx/utr(-/-) mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207.     

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.     

Dystroglycan is not required for localization of dystrophin, syntrophin, and neuronal nitric-oxide synthase at the sarcolemma but regulates integrin alpha 7B expression and caveolin-3 distribution.

Dystroglycan is part of the dystrophin-associated protein complex, which joins laminin in the extracellular matrix to dystrophin within the subsarcolemmal cytoskeleton. We have investigated how mutations in the components of the laminin-dystroglycan-dystrophin axis affect the organization and expression of dystrophin-associated proteins by comparing mice mutant for merosin (alpha(2)-laminin, dy), dystrophin (mdx), and dystroglycan (Dag1) using immunohistochemistry and immunoblots. We report that syntrophin and neuronal nitric-oxide synthase are depleted in muscle fibers lacking both dystrophin and dystroglycan. Some fibers deficient in dystroglycan, however, localize dystrophin at the cell surface at levels similar to that in wild-type muscle. Nevertheless, these fibers have signs of degeneration/regeneration including increased cell surface permeability and central nuclei. In these fibers, syntrophin and nitric-oxide synthase are also localized to the plasma membrane, whereas the sarcoglycan complex is disrupted. These results suggest a mechanism of membrane attachment for dystrophin independent of dystroglycan and that the interaction of sarcoglycans with dystrophin requires dystroglycan. The distribution of caveolin-3, a muscle-specific component of caveolae recently found to bind dystroglycan, was affected in dystroglycan- and dystrophin-deficient mice. We also examined alternative mechanisms of cell-extracellular matrix attachment to elucidate how the muscle basement membrane may subsist in the absence of dystroglycan, and we found the alpha(7B) splice variant of the alpha(7) integrin receptor subunit to be up-regulated. These results support the possibility that alpha(7B) integrin compensates in mediating cell-extracellular matrix attachment but cannot rescue the dystrophic phenotype.     

Integrin alpha3beta1 expressed by human colon cancer cells is a major carrier of oncodevelopmental carbohydrate epitopes

Oncodevelopmental carbohydrate epitopes are a common feature of human colorectal carcinoma, yet their biological significance remains unclear. We have shown previously that monoclonal antibody (MAb) 3A7, which recognizes a determinant on type 2 chain blood group A and B oligosaccharides, detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and some human colon cancer cell lines. (Laferté S et al. [19951 J. Cell. Biochem. 57:101-119). In this study, we set out to purify gp140, the major glycoprotein carrier of the 3A7 epitope expressed by human colon cancer cells, as a first step towards elucidating the contribution of the 3A7 epitope and its major glycoprotein carrier to colon cancer progression. To this end, gp140 was purified from HT29 cells and used for the preparation of polypeptide-specific monoclonal antibodies. Five monoclonal antibodies (7A8, 7B11, 8C7, 8H7, and 11D4) immunoprecipitated a 3A7-immunoreactive glycoprotein complex of 140 kDa which was subsequently identified by partial protein sequencing as alpha3beta1 integrin. Flow cytometric analysis of Chinese hamster ovary (CHO) cells expressing different human integrin chains revealed that MAbs 7A8 and 7B11 detect the alpha3 integrin subunit whereas MAbs 8C7 and 8H7 detect the beta1 integrin subunit. MAb 11D4, which did not bind to any of the CHO transfectants, detected type 2 chain blood group A determinant. Flow cytometric analysis of a panel of human colon carcinoma cell lines obtained from blood group A, AB, or B individuals revealed a direct correlation between cell-surface expression of the 3A7 epitope and alpha3 integrin subunit, suggesting that alpha3beta1 integrin is a preferred target of the 3A7 epitope in colon cancer cells. Using lectins and glycosidases to examine further the carbohydrate structure of alpha3beta1 integrin, we demonstrated that it is a sialoglycoprotein containing both N- and O-linked oligosaccharides. In addition, both alpha3 and beta1 integrin subunits express beta1-6 branched Asn-linked oligosaccharides and short poly-N-acetyllactosamine units (Galbeta1-4GlcNAc-R; n < or = 3), glycans previously implicated in cancer metastasis.Thus, alpha3beta1 integrin expressed by human colon carcinoma cells is a major carrier of oncodevelopmental carbohydrate epitopes whose presence may modulate tumor cell adhesion, migration, and/or invasion.     

The alpha 5 beta 1 integrin associates with a dystrophin-containing lattice during muscle development.

The organization of the alpha 5 beta 1 integrin on skeletal muscle was studied in culture and in sections from adult and embryonic tissue using monoclonal antibodies specific for the alpha 5 subunit. The alpha 5 beta 1 integrin showed changes in organization and in the molecules with which it colocalizes. On early myoblasts, possessing a fibroblast-like morphology, the alpha 5 integrin organization was indistinguishable from that on fibroblasts; it was expressed prominently and localized in numerous focal contacts around the cell periphery. In bipolar myoblasts and early myotubes, the alpha 5 integrin was expressed only weakly and localized in a small number of focal contact-like structures. As myogenesis proceeded there was an apparent increase in integrin expression and a change in organization. In addition to the focal contact-like structures that persist throughout myogenesis in vitro, a dense lattice-like structure of integrin appeared. Fibrillar fibronectin, talin, and non-muscle alpha-actinin did not colocalize with the alpha 5 beta 1 integrin in the lattice structure as they did in the focal contact-like structures. However, dystrophin, which displayed a diffuse distribution earlier, now colocalized with the alpha 5 beta 1 integrin in the punctate lattice. Coincident with the registration of myofibrils into visible sarcomeres, the prominent dense, lattice structure disappeared leaving the focal contact-like structures as the only regions of organized alpha 5 beta 1 integrin. Despite the presence of the beta 1 integrin in neuromuscular or myotendinous junctions in vivo and on myotubes in vitro, the alpha 5 beta 1 integrin was not present in either junction. These observations suggest that the alpha 5 beta 1 integrin is involved in the adhesion of muscle to the extracellular matrix, the organization of the dystrophin-containing lattice, and the organization of nascent myofibrils which emanate from the focal contact- and stress fiber-like structures in muscle. Other integrins appear to anchor myofibrils at the myotendinous and neuromuscular junctions.     

Expression of a beta 1-related integrin by oligodendroglia in primary culture: evidence for a functional role in myelination

We have investigated the expression of integrins by rat oligodendroglia grown in primary culture and the functional role of these proteins in myelinogenesis. Immunochemical analysis, using antibodies to a number of alpha and beta integrin subunits, revealed that oligodendrocytes express only one detectable integrin receptor complex (alpha OL beta OL). This complex is immunoprecipitated by a polyclonal anti-human beta 1 integrin subunit antibody. In contrast, astrocytes, the other major glial cell type in brain, express multiple integrins including alpha 1 beta 1, alpha 3 beta 1, and alpha 5 beta 1 complexes that are immunologically and electrophoretically indistinguishable from integrins expressed by rat fibroblasts. The beta subunit of the oligodendrocyte integrin (beta OL) and rat fibroblast beta 1 have different electrophoretic mobilities in SDS-PAGE. However, the two beta subunits appear to be highly related based on immunological cross- reactivity and one-dimensional peptide mapping. After removal of N- linked carbohydrate chains, beta OL and beta 1 comigrated in SDS-PAGE and peptide maps of the two deglycosylated subunits were identical, suggesting differential glycosylation of beta 1 and beta OL accounts entirely for their size differences. The oligodendrocyte alpha subunit, alpha OL, was not immunoprecipitated by antibodies against well characterized alpha chains which are known to associate with beta 1 (alpha 3, alpha 4, and alpha 5). However, an antibody to alpha 8, a more recently identified integrin subunit, did precipitate two integrin subunits with electrophoretic mobilities in SDS-PAGE identical to alpha OL and beta OL. Functional studies indicated that disruption of oligodendrocyte adhesion to a glial-derived matrix by an RGD-containing synthetic peptide resulted in a substantial decrease in the level of mRNAs for several myelin components including myelin basic protein (MBP), proteolipid protein (PLP), and cyclic nucleotide phosphodiesterase (CNP). These results suggest that integrin-mediated adhesion of oligodendrocytes may trigger signal(s) that induce the expression of myelin genes and thus influence oligodendrocyte differentiation.     

Integrins alpha2beta1 and alpha4beta1 can mediate SA11 rotavirus attachment and entry into cells

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins alpha2beta1 and alpha4beta1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing alpha2beta1 or alpha4beta1 integrins via transfection is increased over virus binding to and infection of cells transfected with alpha3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of alpha2beta1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing alpha2beta1 was elevated over binding to control cells and was specifically blocked by the anti-alpha2 monoclonal antibody AK7. Virus growth in alpha4-transfected K562 cells which had also been induced to express alpha2beta1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, alpha2beta1 and alpha4beta1, are capable of acting as cellular receptors for SA11 rotavirus.     

Antibodies against domain E3 of laminin-1 and integrin alpha 6 subunit perturb branching epithelial morphogenesis of submandibular gland, but by different modes

Branching epithelial morphogenesis requires interactions between the surrounding mesenchyme and the epithelium, as well as interactions between basement membrane components and the epithelium. Embryonic submandibular gland was used to study the roles of two mesenchymal proteins, epimorphin and tenascin-C, as well as the epithelial protein laminin-1 and one of its integrin receptors on branching morphogenesis. Laminin-1 is a heterotrimer composed of an alpha 1 chain and two smaller chains (beta 1 and gamma 1). Immunofluorescence revealed a transient expression of laminin alpha 1 chain in the epithelial basement membrane during early stages of branching morphogenesis. Other laminin-1 chains and alpha 6, beta 1, and beta 4 integrin subunits seemed to be expressed constitutively. Expression of epimorphin, but not tenascin-C, was seen in the mesenchyme during early developmental stages, but a mAb against epimorphin did not perturb branching morphogenesis of this early epithelium. In contrast, inhibition of branching morphogenesis was seen with a mAb against the carboxy terminus of laminin alpha 1 chain, the E3 domain. An inhibition of branching was also seen with a mAb against the integrin alpha 6 subunit. The antibodies against laminin alpha 1 chain and integrin alpha 6 subunit perturbed development in distinct fashions. Whereas treatment with the anti-E3 resulted in discontinuities of the basement membrane at the tips of the branching epithelium, treatment with the mAb against alpha 6 integrin subunit seemed to leave the basement membrane intact. We suggest that the laminin E3 domain is involved in basement membrane formation, whereas alpha 6 beta 1 integrin binding to laminin-1 may elicit differentiation signals to the epithelial cells.     

Potential role of alphav and beta1 integrins as oocyte adhesion molecules during fertilization in pigs

Integrin molecules are cell adhesion molecules that are thought to be involved in sperm-oocyte interaction in rodents and humans. The objective of this study was to evaluate whether integrin molecules were present on the surface of pig oocytes, consistent with involvement in sperm-oocyte interaction in this species. Immunocytochemistry and confocal microscopy were used to evaluate the presence of beta1, and alpha1, alpha2, alpha3, alpha4, alpha5, alpha6 and alphav integrin subunits on the plasma membrane of pig oocytes. The beta1 and alphav integrin subunits were present consistently at the surface of pig oocytes; however, the remaining alpha integrin subunits evaluated were not routinely detected. The antibodies to the beta1 and alphav integrin subunits recognized appropriately sized protein bands on western blots of partially purified oocyte plasma membrane. These two antibodies also recognized oocyte plasma membrane protein isolated from a sperm plasma membrane affinity column. Sperm plasma membrane proteins of 137 and 93 kDa appeared to be the ligands for the beta1 integrin subunit as revealed by a western sandwich blot. Antibody to an extracellular domain of the beta1 integrin subunit reduced pig sperm-oocyte binding (P < 0.05), also indicating an assisting role for a beta1 oocyte integrin subunit in sperm-oocyte interaction in pigs. These results are consistent with an alphavbeta1 pig oocyte integrin interacting with a ligand on the sperm plasma membrane during fertilization.     

Regulation of integrin-type cell adhesion receptors by cytokines.

Integrin heterodimers which share a common beta 1 subunit are the major cellular receptors for many extracellular matrix proteins. Here, we show that two inflammatory mediators, interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), can regulate the expression of the alpha 1 beta 1 integrin heterodimer, known to be a laminin and collagen receptor. In human skin fibroblasts 10 units/ml IL-1 beta increase the biosynthesis of the alpha 1 integrin subunit an average of 4.5-fold. Furthermore, IL-1 beta can turn on alpha 1 subunit expression in MG-63 human osteosarcoma cells even in conditions where the untreated MG-63 cells do not express it in detectable amounts. The effect of TNF-alpha on alpha 1 subunit expression is similar. Both IL-1 beta and TNF-alpha increased MG-63 cell adhesion on laminin. The effect of transforming growth factor-beta 1 (TGF-beta 1) on integrin expression in MG-63 cells has been previously described (Heino, J., and Massagué, J. (1989) J. Biol. Chem. 264, 21806-21811). TGF-beta 1 decreases the biosynthesis of alpha 3 subunit but increases the production of alpha 2 subunit. IL-1 beta potentiates the effects of TGF-beta 1. Furthermore, in the presence of TGF-beta 1 the increase in the expression of alpha 1 subunit by IL-1 beta is even larger. Thus, IL-1 beta and TGF-beta 1, which usually have antagonistic functions in connective tissue, can regulate integrin expression in a synergistic way.     

Laminin-binding integrin alpha 7 beta 1: functional characterization and expression in normal and malignant melanocytes.

A novel integrin, alpha 7 beta 1, that specifically binds with high affinity to laminin has been identified on melanoma cells. This complex was purified from both human and murine melanoma cells by laminin-affinity chromatography, and the alpha 7 subunit was recovered after gel electrophoresis. N-terminal amino acid sequence analysis of the alpha 7 subunit from both human and mouse cells verifies that this integrin is distinct from other alpha chains in the beta 1 family, although strikingly similar to the alpha 6 subunit. By using specific proteolytically derived fragments of laminin, it was determined that the alpha 7 beta 1 complex binds selectively to the E8 region, which represents part of the long arm of laminin. In contrast, the receptor failed to bind to the P1 fragment, which contains the intersection of the short arms of laminin. Although the alpha 7 beta 1 complex was commonly expressed in melanoma cells, this integrin was not detected in normal melanocytes, suggesting that alpha 7 expression may be associated with malignant transformation. These results establish the existence of a novel integrin that binds to the E8 domain of laminin and appears to mediate cell adhesion to this ligand.     

Integrin alpha3 subunit participates in myoblast adhesion and fusion in vitro

Satellite cells are myogenic precursor cells, participating in growth, and regeneration of skeletal muscles. The proteins that play a role in myogenesis are integrins. In this report, we show that the integrin alpha3 subunit is expressed in quiescent satellite cells and activated myoblasts. We also find that in myoblasts the integrin alpha3 subunit is localized at cell-cell and cell-extracellular matrix contacts. We notice that increase in protein and mRNA encoding the integrin alpha3 subunit accompanies myoblast differentiation. Using double immunofluorescence and immunoprecipitation experiments, we demonstrate that the integrin alpha3 subunit co-localizes with actin, and binds the integrin beta1 subunit and ADAM12, suggesting that the complex alpha3beta1/ADAM12 is probably involved in myoblast fusion. Importantly, overexpression of the full-length integrin alpha3 subunit increases myoblast fusion whereas an antibody against its extracellular domain inhibits fusion. These data demonstrate that the integrin alpha3 subunit may contribute to satellite cell activation and then myoblast adhesion and fusion.     

Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains

The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits. The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.     

Cib2 binds integrin alpha7Bbeta1D and is reduced in laminin alpha2 chain-deficient muscular dystrophy

Mutations in the gene encoding laminin alpha2 chain cause congenital muscular dystrophy type 1A. In skeletal muscle, laminin alpha2 chain binds at least two receptor complexes: the dystrophin-glycoprotein complex and integrin alpha7beta1. To gain insight into the molecular mechanisms underlying this disorder, we performed gene expression profiling of laminin alpha2 chain-deficient mouse limb muscle. One of the down-regulated genes encodes a protein called Cib2 (calcium- and integrin-binding protein 2) whose expression and function is unknown. However, the closely related Cib1 has been reported to bind integrin alphaIIb and may be involved in outside-in-signaling in platelets. Since Cib2 might be a novel integrin alpha7beta1-binding protein in muscle, we have studied Cib2 expression in the developing and adult mouse. Cib2 mRNA is mainly expressed in the developing central nervous system and in developing and adult skeletal muscle. In skeletal muscle, Cib2 colocalizes with the integrin alpha7B subunit at the sarcolemma and at the neuromuscular and myotendinous junctions. Finally, we demonstrate that Cib2 is a calcium-binding protein that interacts with integrin alpha7Bbeta1D. Thus, our data suggest a role for Cib2 as a cytoplasmic effector of integrin alpha7Bbeta1D signaling in skeletal muscle.     

Identification of a neuronal laminin receptor: an Mr 200K/120K integrin heterodimer that binds laminin in a divalent cation-dependent manner

Neuronal interactions with extracellular matrix (ECM) components are crucial for axon growth and guidance during development and nerve regeneration. Laminin (LN), a prominent ECM glycoprotein, promotes neuronal survival and axon growth. To identify neuronal receptors for LN, we looked for cell surface proteins on the neuronal cell line B50 that bind LN. An integrin alpha/beta 1 dimeric receptor was identified and purified using lectin and LN affinity chromatography. The purified integrin contains two subunits with Mrs of 200 K and 120 K that bind LN specifically in the presence, but not the absence, of divalent cations (Ca2+/Mg2+ or Ca2+/Mn2+). The Mr 120 K protein was identified as the rat integrin beta 1 subunit using two beta 1 subunit-specific antibodies, and was shown to form a noncovalent complex with the Mr 200K putative alpha subunit. Since neurons and neuronal cell lines express similar integrin beta 1-class heterodimers that mediate attachment and process outgrowth on LN, the Mr 200K/120K complex identified here is likely to be an important laminin receptor used by neurons. This integrin may also mediate binding to LN by many nonneuronal cell types.     

Integrin alpha 6 beta 4 forms a complex with the cytoskeletal protein HD1 and induces its redistribution in transfected COS-7 cells.

The integrin alpha 6 beta 4 is a major component of hemidesmosomes, in which it is linked to intermediate filaments. Its presence in these structures is dependent on the beta 4 cytoplasmic domain but it is not known whether beta 4 interacts directly with keratin filaments or by interaction with other proteins. In this study, we have investigated the interaction of GST-cyto beta 4A fusion proteins with cellular proteins and demonstrate that a fragment of beta 4A, consisting of the two pairs of fibronectin type III repeats, separated by the connecting segment, forms a specific complex containing a 500-kDa protein that comigrates with HD1, a hemidesmosomal plaque protein. A similar protein was also bound by a glutathione S-transferase fusion protein containing the cytoplasmic domain of a variant beta 4 subunit (beta 4B), in which a stretch of 53 amino acids is inserted in the connecting segment. Subsequent immunoblot analysis revealed that the 500-kDa protein is in fact HD1. In COS-7 cells, which do not express alpha 6 beta 4 or the hemidesmosomal components BP230 and BP180, HD1 is associated with the cytoskeleton, but after transfecting the cells with cDNAs for human alpha 6 and beta 4, it was, instead, colocalized with alpha 6 beta 4 at the basal side of the cells. The organization of the vimentin, keratin, actin, and tubulin cytoskeletal networks was not affected by the expression of alpha 6 beta 4 in COS-7 cells. The localization of HD1 at the basal side of the cells depends on the same region of beta 4 that forms a complex containing HD1 in vitro, since the expression of alpha 6 with a mutant beta 4 subunit that lacks the four fibronectin type III repeats and the connecting segment did not alter the distribution of HD1. The results indicate that for association of alpha 6 beta 4 with HD1, the cytoplasmic domain of beta 4 is essential. We suggest that this association may be crucial for hemidesmosome assembly.     

Extraocular muscle is spared upon complete laminin alpha2 chain deficiency: comparative expression of laminin and integrin isoforms.

Mutations in the gene encoding laminin (LM) alpha2 chain cause congenital muscular dystrophy. Here, we show that extraocular muscle (EOM) is spared upon complete LMalpha2 chain absence. The major LM chains in limb muscle basement membranes are alpha2, beta1, beta2 and gamma1 whereas alpha2, alpha4, beta1, beta2 and gamma1 chains are expressed in EOM. Expression of LMalpha4 chain mRNA is further increased in LMalpha2 chain deficient EOM. Mainly integrin alpha7X1 subunit, which binds to laminin-411, is expressed in EOM and in contrast to dystrophic limb muscle, sustained integrin alpha7B expression is seen in LMalpha2 chain deficient EOM. We propose that LMalpha4 chain, possibly by binding to integrin alpha7BX1beta1D, protects EOM in LMalpha2 chain deficient muscular dystrophy.