Identification of the mouse muscle 43,000-dalton acetylcholine receptor-associated protein (RAPsyn) by cDNA cloning

The nicotinic acetylcholine receptor and a receptor-associated protein of 43 kDa are the major proteins present in postsynaptic membranes isolated from Torpedo electric organ. Immunochemical analyses indicated that a protein sharing antigenic determinants with the receptor-associated protein is also present at receptor clusters of muscle cell lines and postsynaptic membranes of vertebrate neuromuscular junctions. We now provide definitive proof that a homolog of the 43-kDa protein exists in mammals. Complimentary DNA clones encoding the complete protein sequence have been isolated from the mouse muscle cell line, BC3H1. We heretofore refer to these proteins as nicotinic receptor-associated proteins at synapses or N-RAP-syns. The deduced sequence of mouse RAPsyn has 412 amino acids and a molecular mass of 46,392 daltons. The overall identity with Torpedo RAPsyn is 70%; some regions are extremely well conserved and are therefore postulated to be functionally important. Important domains, including the amino terminus and a cAMP-dependent protein kinase phosphorylation site, are conserved between species. Several structural features are consistent with the proposal that RAPsyn is a peripheral membrane protein that associates with membranes by virtue of covalently bound myristate. Although multiple mRNAs were previously identified in Torpedo electric organ, RNA blot analysis reveals a single polyadenylated RAPsyn mRNA of approximately equal to 2.0 kilobases in newborn and 4-week-old mouse muscle. Finally, genomic DNA blot analysis indicates that a single N-RAPsyn gene is present in the mouse genome.