Agrin-like molecules at synaptic sites in normal, denervated, and damaged skeletal muscles.

Abstract

Abstract. Several lines of evidence have led to the hypothesis that agrin, a protein extracted from the electric organ of Torpedo, is similar to the molecules in the synaptic cleft basal lamina at the neuromuscular junction that direct the formation of acetylcholine receptor and acetylcholinesterase aggregates on regenerating myofibers. One such finding is that monoclonal antibodies against agrin stain molecules concentrated in the synaptic cleft of neuromuscular junctions in rays. In the studies described here we made additional monoclonal antibodies against agrin and used them to extend our knowledge of agrin-like molecules at the neuromuscular junction. We found that antiagrin antibodies intensely stained the synaptic cleft of frog and chicken as well as that of rays, that denervation of frog muscle resulted in a reduction in staining at the neuromuscular junction, and that the synaptic basal lamina in frog could be stained weeks after degeneration of all cellul~ir components of the neuromuscular junction. We also describe anti-agrin staining in nonjunctional regions of muscle. We conclude the following: (a) agrin-like molecules are likely to be common to all vertebrate neuromuscular junctions; (b) the long-term maintenance of such molecules at the junction is nerve dependent; (c) the molecules are, indeed, a component of the synaptic basal lamina; and (d) they, like the molecules that direct the formation of receptor and esterase aggregates on regenerating myofibers, remain associated with the synaptic basal lamina after muscle damage.

The agrin gene codes for a family of basal lamina proteins that differ in function and distribution

“…Immunohistochemical studies (Reist et al, 1987;Magill-Solc and McMahan, 1988;Godfrey et al, 1988b;Fallon and Gelfman, 1989) on muscles and nerves in chick embryos have demonstrated that, in addition to staining the synaptic portion of the muscle fiber's basal lamina, antibodies directed against agrin stain the basal lamina of the Schwann cells that cap the axon terminal at the neuromuscular junction and ensheath the preterminal axons within nerves. The basal lamina of muscle fibers stains more or less uniformly prior to innervation (Godfrey et al, 1988b), and such staining persists in the embryo even if innervation is prevented (FaIIon and Gelfman, 1989).…”

“…In addition to staining nonsynaptic basal lamina in skeletal muscle, nerve, and CNS tissues, anti-agrin MAbs stain the glomerular basal lamina in the chick kidney (Godfrey, 1991), the myofiber basal lamina in cardiac muscle, the basal lamina of smooth muscle cells, and tunica intima in certain blood vessels (Reist et al, 1987;Godfrey et al, 1988a). Neither AChRs nor AChE is aggregated in the plasma membranes of the cells with wh ich these basal laminae are associated.…”

“…Nonetheless, the protein secreted from the transfected COS-7 cells in all cases was recognized by anti-agrin MAb 5B1 (see below), which was raised against Torpedo agrin and has been shown to stain the synaptic basal lamina at neuromuscular junctions in chick (Reist et al, 1987).…”

“…The medium was broughtto 150 mM NaCI, 5 mM EDTA, 1% Triton X-100, 50 mM Tris-HCI (pH 7.5). and 5 !ll of aseites fluid containing the MAb 5B1 (Reist et al, 1987) was added. After overnight incubation at 4°C, 100 !ll of a 1:1 suspension of protein A-Sepharose beads (Pharmacia) was added, and the incubation was continued for 2 hr at room temperature.…”

“…However, basal lamina at nonsynaptic sites in a variety of tissues including muscle, central nervous system (CNS), heart, and kidney, where AChRs and AChE are not known to aggregate, is also stained by these antibodies (Reist et al, 1987;Godfrey et al, 1988aGodfrey et al, , 1988b. Moreover, proteins inactive in AChR aggregation assays are immunoprecipitated from extracts of several different tissues by antiagrin MAbs (Nitkin et al, 1987;Godfrey, 1991).…”