Synaptic Integrins in Developing, Adult, and Mutant Muscle: Selective Association of α1, α7A, and α7B Integrins with the Neuromuscular Junction

Abstract

Differentiation of both pre- and postsynaptic structures at the skeletal neuromuscular junction is organized by the basal lamina that occupies the synaptic cleft. As beta1 integrins are a major class of receptors for basal lamina components, we stained muscles with antibodies to the 10 integrin alpha subunits known to form dimers with beta1, to determine if any of these molecules were concentrated at synaptic sites on muscle fibers. In both developing and adult muscle, the integrin alpha1 chain was selectively associated with presynaptic cells (Schwann cells and/or nerve terminals), while alpha7 was present on both synaptic and extrasynaptic portions of the muscle fiber surface. Thus alpha1 and alpha7 integrins are present in synaptic membranes. Expression of the alpha7 chain was analyzed further by staining with antibodies specific for three alternatively spliced products of the alpha7 gene (A, B, and C), all of which were expressed in muscle. The alpha7A and alpha7B isoforms were confined to synaptic sites in adult muscle, while alpha7C was present both synaptically and extrasynaptically. In developing muscle, alpha7A appeared postnatally and specifically at the synapse; alpha7B was present throughout the muscle fiber perinatally, becoming confined to the synapse in the second postnatal week; and alpha7C was present extrasynaptically both perinatally and in adulthood. Thus, two of the alpha7 integrins are synapse-specific, and all three show distinct spatiotemporal patterns of expression within a single cell type. Finally, we asked whether perturbation of laminin expression affected the distribution of the alpha7 integrins. In normal mice, laminin beta2 is concentrated in synaptic basal lamina. In beta2-null mutant mice, alpha7A was still present at synaptic sites, but alpha7B was absent. This result provides genetic evidence that basal lamina composition is a determinant of integrin distribution.

Expression of muscle-specific integrins in masseter muscle fibers during malocclusion disease

“…In particular, the α7β1-integrin is concentrated at neuromuscular and myotendinous junctions and it is located along the sarcolemma at costameres with an important role in the functions of the skeletal muscle (13). It was demonstrated that congenital myopathies may be caused by mutations in the human integrin α7 gene (ITGA7) confirming the importance of the α7β1-integrin in maintaining normal skeletal muscle physiology (14).…”

“…Regarding the α7 subunit, the α7B isoform is detected in proliferating and adult myofibres and is exclusively localized in the neuromuscular and myotendinous junctions (13,15). Instead, the α7A isoform, detected in differentiating myofibres, plays a key role in muscle regeneration during the dynamic adhesion stage, whereas it appears to have a minor role in mature skeletal muscle (16).…”

Myotendinous Junction Defects and Reduced Force Transmission in Mice that Lack α7 Integrin and Utrophin

“…22,23 At least six ␣7 integrin isoforms produced by developmentally regulated RNA splicing are expressed in skeletal muscle. 24 Mutations in the ␣7 integrin gene (ITGA7) cause myopathy in humans.…”

The β1 Cytoplasmic Domain Regulates the Laminin-binding Specificity of the α7X1 Integrin

“…Samples including controls for whole cell lysates (10 g protein/lane) were processed for SDS-PAGE under nonreducing conditions and then transferred to Immobilon-P membranes (Millipore, Bedford, MA). The membranes were incubated with rabbit antibody against human integrin ␤1 (pAb 22778) (Martin et al, 1996;Yao et al, 1997) or mouse ␤1 (mAb 2000; Chemicon International). In a different approach to assess the ␣7 preference to ␤1A or ␤1D, equal amounts of cell lysate (500 g of protein) prepared from X1/D-27 cells (a clone of X1/D cells) were immunoprecipitated with human ␤1A or mouse ␤1D antibody for three consecutive rounds.…”

“…The laminin-binding ␣7␤1 integrin, expressed in mouse skeletal muscle as early as E10.5 d of development, mediates myoblast motility on laminin substrates (Kaufman et al, 1980;Kaufman et al, 1991;Yao et al, 1996a,b;Crawley et al, 1997;von der Mark et al, 2002). In mature skeletal muscle, the ␣7 receptor is associated with costameres and the myotendinous and neuromuscular junctions (Bao et al, 1993;Belkin et al, 1996;Martin et al, 1996;van der Flier et al, 1997). The physiological importance of ␣7 is demonstrated in ␣7-null mutant mice, which develop a form of muscular dystrophy with myotendinous junction (MTJ) defects (Mayer et al, 1997), and in humans with mutations in ␣7 gene, which develop congenital myopathies (Hayashi et al, 1998;Pegoraro et al, 2002).…”