ARIA, a protein that stimulates acetylcholine receptor synthesis, also induces tyrosine phosphorylation of a 185-kDa muscle transmembrane protein.

Abstract

Motoneurons promote the accumulation of acetylcholine receptors (AChRs) at developing neuromuscular synapses. The AChR-mdudng activity protein ARIA, which is purified from chicken brain and increases the synthesis of AChRs in chicken myotubes, may play a crucial role in this process. Here we show that ARIA induces the rapid tyrosine phosphorylation of a Mr 185,000 protein (p185) in muscle cells. Phosphorylation of p185 correlates with AChR induction at each stage of ARIA purification. Moreover, medium conditioned by spinal cord motoneurons stimulates AChR synthesis and p185 phosphorylation. Studies with membrane-impermeant reagents and 125I-labeled ARIA indicate that p185 is a transmembrane ARIA-receptor tyrosine kinase. Our data suggests that muscle AChR synthesis can be regulated through tyrosine phosphorylation.During formation of the neuromuscular junction, embryonic motor nerve terminals promote the accumulation of acetylcholine receptors (AChRs) and other synaptic proteins in the postsynaptic muscle membrane and cytoplasm. The increase in postsynaptic AChR density is due primarily to an increase in local AChR synthesis (1) and to the aggregation of receptors already present in the membrane at the time of nervemuscle contact (2). There is strong evidence that a form ofthe protein agrin plays a role in AChR aggregation (3,4). Less is known about factors that induce AChR gene expression. We have purified a 42-kDa glycoprotein from chicken brain that stimulates AChR synthesis in cultured embryonic myotubes (5,6). Our hypothesis is that this protein, named ARIA for its acetylcholine receptor inducing activity, regulates receptor synthesis at developing neuromuscular junctions. ARIA stimulation of AChR synthesis is associated with an increase in the level of the a-subunit mRNA in chicken muscle (7). In mouse muscle a prominent effect on the E-subunit mRNA was observed (8). The local increase in AChR synthesis is remarkable because it occurs despite a dramatic decrease in AChR synthesis in extrasynaptic regions of the same muscle cells, as synaptic transmission drives muscle electrical/ mechanical activity.We have begun to study the mechanism by which ARIA regulates AChR subunit gene expression. In this paper we present evidence suggesting that ARIA regulates muscle AChR synthesis by activating a receptor tyrosine kinase. MATERIALS AND METHODSTissue Culture. Chicken myoblasts were dissociated from embryonic day 11 (Eli) pectoral muscle and seeded in 96-or 48-well culture dishes (20,000 and 80,000 cells per well, respectively) in Eagle's minimum essential medium (MEM)/ 10%o horse serum/2% chicken embryo extract. L6 cells were grown in Dulbecco's MEM (DMEM)/10% fetal bovine serum. Motoneurons were dissociated from chicken spinal cords (E5) and purified through metrizamide gradients (9) before plating. Cells were plated in DMEM/F-12 (GIBCO), supplemented with glucose to 4 g/liter, 10%o fetal bovine serum, and chicken muscle extract at 10 ,ug/ml (9).ARIA Purification. ARIA was purified by a modification of ou...

Endogenous Neuregulin Restores Radial Glia in a (Ferret) Model of Cortical Dysplasia

“…The activity of the fractions was assessed on L6 rat myoblast cells by analysis of the induction of p185 tyrosine phosphorylation, as described by Corfas et al (1993). Briefly, cells were grown in DMEM containing 10% fetal bovine serum, Pen/Strep, and L-glutamine.…”

Erbin Is a Protein Concentrated at Postsynaptic Membranes That Interacts with PSD-95

“…In fact, one initially identified isoform of neuregulin is ARIA (for acetylcholine receptor inducing activity) (4). Neuregulin is synthesized in motoneurons (4) released from motoneurons and deposited in the synaptic cleft (49,50), activates ErbB receptors (3,6,51) and subsequent multiple signaling pathways (6,9,52,53) in the skeletal muscle to increase acetylcholine receptor expression. Results from studies of neuregulin-1 and ErbB2 mutant mice indicate that neuregulin is essential for the formation and maintenance of the neuromuscular junction (43,54).…”

The Cellular Response to Neuregulins Is Governed by Complex Interactions of the erbB Receptor Family

“…NRG also modulates the differentiation and proliferation of neuroectodermal cells. NRGs act as glial cell growth factors (28), may specify a glial cell fate for neural crest stem cells (45), appear to mediate axon-induced mitogenesis of Schwann cells (32), and stimulate acetylcholine receptor synthesis at neuromuscular junctions (5,12,20). Furthermore, NRG expression patterns suggest important functions in neurogenesis and in mesenchymal-epithelial cell interactions during development (6,28,30,33).…”