Synapse-Associated Expression of an Acetylcholine Receptor-Inducing Protein, ARIA/Heregulin, and Its Putative Receptors, ErbB2 and ErbB3, in Developing Mammalian Muscle

Abstract

Developing motor axons induce synaptic specializations in muscle fibers, including preferential transcription of acetylcholine receptor (AChR) subunit genes by subsynaptic nuclei. One candidate nerve-derived signaling molecule is AChR-inducing activity (ARIA)/heregulin, a ligand of the erbB family of receptor tyrosine kinases. Here, we asked whether ARIA and erbB kinases are expressed in patterns compatible with their proposed signaling roles. In developing muscle, ARIA was present not only at synaptic sites, but also in extrasynaptic regions of the muscle fiber. ARIA was synthesized, rather than merely taken up, by muscle cells, as indicated by the presence of ARIA mRNA in muscle and of ARIA protein in a clonal muscle cell line. ARIA-responsive myotubes expressed both erbB2 and erbB3, but little EGFR/erbB1 or erbB4. In adults, erbB2 and erbB3 were localized to the postsynaptic membrane. ErbB3 was restricted to the postsynaptic membrane perinatally, at a time when ARIA was still broadly distributed. Thus, our data are consistent with a model in which ARIA interacts with erbB kinases on the muscle cell surface to provide a local signal that induces synaptic expression of AChR genes. However, much of the ARIA is produced by muscle, not nerve, and the spatially restricted response may result from the localization of erbB kinases as well as of ARIA. Finally, we show that erbB3 is not concentrated at synaptic sites in mutant mice that lack rapsyn, a cytoskeletal protein required for AChR clustering, suggesting that pathways for synaptic AChR expression and clustering interact.

The EGF‐like domain of chick acetylcholine receptor‐inducing activity (ARIA) contains its full biological activity

“…Application of ARIAsi36-K205-Fc induced the phosphorylation of erbB 3 receptor in C2C12 myotube cultures (Fig. 4), that was similar to the activity of full-length recombinant ARIA previously described [6,15,23]. Both ARIA and ARIAsi36_K205-Fc induced tyrosine phosphorylation of a protein with a molecular mass of -185 kDa.…”

“…In the local synthesis of post-synaptic molecules, RNAs encoding AChR subunits, AChE and other synapse specific proteins are highly concentrated in the synaptic regions [4][5][6][7]. The up-regulation of AChR synthesis at the neuromuscular junctions is induced by motor neurons.…”

Expression of Neuregulins and their Putative Receptors, ErbB2 and ErbB3, Is Induced during Wallerian Degeneration

“…It is conceivable that Schwann cells may be initially influenced by ␤NRGs liberated from the degenerating axon, with subsequent proliferation and /or other effects (see below) mediated by ␣-and /or ␤NRGs produced by the Schwann cells themselves. This potential for dual sources of NRG is reminiscent of recent observations in skeletal muscle that suggest that erbB receptors at the neuromuscular synapse may be activated either by ␤NRG secreted by peripheral nerve or a mixture of ␣-and ␤NRGs produced by the muscle fiber itself (Moscoso et al, 1995).…”

“…We and others have not detected NRG immunoreactivity in more proximal axonal segments in noninjured nerve. However, there is evidence that NRGs synthesized in the neuronal cell body are transported to the most distal portions of the axon Jo et al, 1995;Moscoso et al, 1995;Sandrock et al, 1995). This suggests that levels of NRG protein that are below the limits of detection immunohistochemically may be present in the more proximal segments of noninjured axons.…”

“…Therefore, we used RT-PCR with axotomized sciatic nerve, DRG and spinal cord templates to isolate partial cDNAs of the rat EGF, erbB3, and erbB4 receptors, and sequenced these clones to establish their relationship to the human genes. Each of the rat clones was found to be closely related to their human and (when sequence was available) mouse orthologs (Luetteke et al, 1994;Moscoso et al, 1995). The rat erbB3 and erbB4 clones were used to screen the nerve and spinal cord libraries.…”