Α7β1 Integrin Research Articles

A functional role for specific spliced variants of the alpha7beta1 integrin in acetylcholine receptor clustering.

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the alpha7beta1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, alpha7A and alpha7B, and the extracellular spliced forms, alpha7X1 and alpha7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the alpha7beta1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-alpha7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-alpha7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the alpha7A and alpha7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the alpha7X2 extracellular domain were active. These results demonstrate that the alpha7beta1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the alpha7 chain, and that laminin, agrin, and the alpha7beta1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.

Interaction of Integrin α7β1 in C2C12 Myotubes and in Solution with Laminin

The dimer of integrin α7 and β1 is a major laminin-binding receptor in skeletal muscle. We studied interactions of integrin α7β1 with the extracellular matrix protein laminin in solution and in intact cells. Integrin α7β1 bound to EHS laminin (laminin-1, composed of α1, β1, and γ1 chains), but not to endogenous laminin expressed in C2C12 myotubes. Northern blot analysis demonstrated that C2C12 myotubes synthesized laminin-1 α, β, and γ subunits mRNAs. C2C12 laminin was, however, immunologically distinct from EHS laminin; it was not recognized by 5D3 anti-laminin-1 monoclonal antibody, whereas 5A2 and LT3 antibodies reacted equally well with C2C12 and EHS laminins. Following deglycosylation of EHS laminin, separation of the subunits by SDS–PAGE, Western blotting, and partial amino acid sequencing of the protein bands, the epitope recognized by 5D3 antibody was localized to the γ1 laminin chain. Following bindingin vitro,the complex of EHS laminin and integrin α7β1 was subject to chemical cross-linking. The two proteins did not undergo cross-linking at the cell surface, consistent with the fact that in intact, resting myotubes integrin α7β1 interacted poorly with EHS laminin, which may reflect a limited accessibility of integrin α7β1 in the membrane to laminin or an inactive state of the integrin.

A Novel Extracellular Domain Variant of the Human Integrin α7 Subunit Generated by Alternative Intron Splicing

The integrin α7β1 laminin receptor, which is expressed on replicating myoblasts, and upregulated during myogenic differentiation, is involved in cell adhesion and communication between muscle cells and the extracellular matrix. It is a major cell-surface substrate in skeletal muscle cells for the cell-surface, arginine-specific, ADP-ribosyltransferase. Both the extracellular and cytoplasmic domains of the mouse α7 subunit undergo alternative splicing during development, generating differentially expressed variants with presumably unique ligand-binding and signalling properties. Here human cDNA clones isolated from a fetal heart λgt10 cDNA library encoded the complete sequence of the α7 subunit and hybridised to a single major 4.4 kb α7 subunit transcript abundantly expressed in human skeletal muscle, moderately expressed in heart, and weakly expressed in most other tissues. One clone out of four contained a novel 225-nucleotide in-frame deletion corresponding to 75 amino acids in the C-terminal region of the extracellular domain. The variant, whose expression appears to be tissue-specific, is created by alternative splicing at sites flanking an intron in the α7 gene. A related mouse form was identified in P19 embryonal carcinoma cells. Deletion of the spliced region, which either contains or is in very close proximity to the major ADP-ribosylation site of the α7 subunit, may serve to modulate the effects of ADP-ribosylation, or alternatively molecular associations, and receptor-ligand affinity.

Altered expression of the alpha7beta1 integrin in human and murine muscular dystrophies.

The alpha7beta1 integrin is the primary laminin receptor on skeletal myoblasts and adult myofibers. It has distinct functions during muscle development and contributes to muscle structural integrity. We have studied this integrin in cases where expression of dystrophin or laminin are compromised. Immunofluorescence demonstrates an increase in alpha7beta1 in patients with Duchenne muscular dystrophy and in mdx mice that lack dystrophin. Analysis of RNA from mdx mice and from patients with Duchenne and Becker muscular dystrophies indicates that the increase in the alpha7beta1 integrin is regulated at the level of alpha7 gene transcription. In contrast, the levels of alpha7beta1 integrin are severely diminished in patients with laminin alpha2 chain congenital dystrophy muscular dystrophy and in dy/dy mice that also do not make the alpha2 laminin chain. Analysis of RNA from the hindlimbs of dy/dy mice demonstrated that in the absence of laminin alpha7 gene transcription is inhibited and limited to specific alternatively spliced isoforms. We suggest that the increased expression of alpha7beta1 integrin in the absence of dystrophin compensates for the reduced dystrophin-mediated linkage of fibers with the basal lamina and modulates the development of pathology associated with these diseases. The decrease in alpha7beta1 integrin and its transcripts in the absence of laminin likely contributes to the severe myopathy that results from laminin alpha2 chain deficiency and suggests that laminin-2 regulates expression of the alpha7 integrin gene. The role of the alpha7beta1 integrin in muscle integrity also suggests that compromised expression of this receptor may underlie as yet undefined myopathies.

The α7β1 Integrin Mediates Adhesion and Migration of Skeletal Myoblasts on Laminin

Many aspects of myogenesis are believed to be regulated by myoblast interactions with specific components of the extracellular matrix. For example, laminin has been found to promote adhesion, migration, and proliferation of mammalian myoblasts. Based on affinity chromatography, the α7β1 integrin has been presumed to be the major receptor mediating myoblast interactions with laminin. We have prepared a monoclonal antibody, O26, that specifically reacts with both the X1 and the X2 extracellular splice variants of the α7 integrin chain. This antibody completely and selectively blocks adhesion and migration of rat L8E63 myoblasts on laminin-1, but not on fibronectin. In contrast, a polyclonal antibody to the fibronectin receptor, α5β1 integrin, blocks myoblast adhesion on fibronectin, but not on laminin-1. The α7β1 integrin also binds to a mixture of laminin-2 and laminin-4, the major laminin isoforms in developing and adult skeletal muscle, but O26 is a much less potent inhibitor of myoblast adhesion on the laminin-2/4 mixture than on laminin-1. Based on affinity chromatography, we suggest that this may be due to higher affinity binding of α7X1 to laminin-2/4 than to laminin-1.

Transforming growth factor-beta regulates collagen gel contraction by increasing alpha 2 beta 1 integrin expression in osteogenic cells.

The contraction of floating collagen gels is suggested to mimic the reorganization of collagenous matrix during development and tissue healing. Here, we have studied two osteogenic cell lines, namely MG-63 and HOS, and a chemically transformed subclone of HOS cells, HOS-MNNG. Transforming growth factor-beta (TGF-beta), a putative regulator of bone fracture healing, increased collagen gel contraction by MG-63 and HOS-MNNG, but not by HOS cells. Our data show that TGF-beta-induced fibronectin synthesis is not sufficient for the process. Instead, anti-beta 1 integrin antibodies could prevent the contraction. There are three different integrin heterodimers that are known to mediate the cell-collagen interaction, namely alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1. In MG-63 cells TGF-beta increased the expression of alpha 2 beta 1 integrin and decreased the expression of alpha 3 beta 1 integrin, whereas alpha 1 beta 1 integrin is not expressed. HOS cells had no alpha 2 beta 1 integrin, neither did TGF-beta induce its expression. However, HOS-MNNG cells expressed more alpha 2 beta 1 integrin when treated with TGF-beta. Thus, we suggest that the mechanism of the enhanced collagen gel contraction by TGF-beta is the increased expression of alpha 2 beta 1 integrin heterodimer. To further test this hypothesis, we expressed a full-length alpha 2 integrin cDNA in HOS cells and in MG-63 cells. We obtained HOS cell clones that expressed alpha 2 beta 1 heterodimer, and the ability of these cells to contract collagen gels was greatly enhanced. Furthermore, the contraction by MG-63 cells transfected with alpha 2 integrin cDNA was enhanced, and the contraction by cells transfected with antisense oriented alpha 2 integrin cDNA was decreased. Thus, both in MG-63 and HOS cells the increased alpha 2 integrin expression alone was sufficient for the enhanced contraction of collagen gels. Furthermore, the amount of alpha 2 integrin is critical for the process, and its decrease leads to diminished ability to contract gels.

Selective modulation of the interaction of α7β1 integrin with fibronectin and laminin by l-14 lectin during skeletal muscle differentiation

The alpha 7 beta 1 integrin was originally identified and isolated from differentiating skeletal muscle and shown to be a laminin-binding protein (Song et al. (1992) J. Cell Biol. 117, 643-657). Expression of the alpha 7 gene and protein are developmentally regulated during skeletal muscle differentiation and have been used to identify cells at distinct stages of the myogenic lineage (George-Weinstein et al. (1993) Dev. Biol. 156, 209-229). The lactoside-binding protein L-14 exists as a dimer and has been localized on a variety of cells, in association with extracellular matrix. During myogenesis in vitro, L-14 is synthesized within replicating myoblasts but it is not secreted until these cells commence terminal differentiation and fusion into multinucleate fibers (Cooper and Barondes, J. Cell Biol. (1990) 110, 1681-1691). Addition of purified L-14 to myogenic cells plated on laminin inhibits myoblast spreading and fusion, suggesting that the L-14 lectin regulates muscle cell interactions with the extracellular matrix that are germane to myogenic development (Cooper et al. (1991) J. Cell Biol. 115, 1437-1448). We demonstrate here, using affinity chromatography and immunoblots, that alpha 7 beta 1 also binds to fibronectin and to the L-14 lectin. L-14 binds to both laminin and to the alpha 7 beta 1 integrin, and it can effectively inhibit the association of laminin and this integrin. Modulation of alpha 7 beta 1 interaction with its ligands by L-14 is selective: L-14 does not bind to fibronectin, nor does it interfere with the binding of fibronectin to alpha 7 beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha 7 beta 1 integrin is a component of the myotendinous junction on skeletal muscle.

Immunization against a 70 kDa band that co-purifies with skeletal muscle integrins has resulted in an antibody directed against the avain alpha 7 integrin subunit. The specificity of the antibody was established by patterns of tissue staining and cross-reactivity with antibodies directed against the cytoplasmic domain of the rat alpha 7 cytoplasmic domain. On sections of adult skeletal muscle the alpha 7 integrin was enriched in the myotendinous junction (MTJ). This localization was unique as neither the alpha 1, alpha 3, alpha 5, alpha 6 and alpha v subunit localizes in the myotendinous junction. The distribution of the alpha 7 subunit in the MTJ was examined during embryonic development. alpha 7 expression in the junction is first apparent around embryo day 14 and is almost exclusively at the developing MTJ at this stage. alpha 3 is expressed with distinctive punctate staining around the junctional area in earlier embryos (11-day). The time of appearance of the alpha 7 subunit in the MTJ correlates with the insertion of myofibrils into subsarcolemmal densities and folding of the junctional membrane, suggesting a role of the alpha 7 integrin in this process. Vinculin is present throughout development of the myotendinous junction, suggesting that the alpha 7 integrin recognizes a preformed cytoskeletal structure. The presence of the alpha 7 subunit in the myotendinous junction and the alpha 5 subunit in the adhesion plaque demonstrates a molecular difference between these two adherens junctions. It also points to possible origins of junctional specificity on muscle. Differences between these two junctions were developed further using an antibody against phosphotyrosine (PY20). Phosphotyrosine is thought to participate in the organization and stabilization of adhesions. The focal adhesion and the neuromuscular junction, but not the MTJ, contained proteins phosphorylated on tyrosine.