Although the metabolic half-life of muscle endplate acetylcholine receptor (AChR) changes during development and after denervation in the adult, little is known about the molecular mechanisms that influence receptor stability. We have investigated the effect on AChR turnover of its interaction with rapsyn, a 43 kDa peripheral membrane protein that is closely associated with the AChR in muscle cells and is required for its clustering at endplates. Both in transfected COS cells and in cultured myotubes from rapsyn-negative and rapsyn-positive mice, we have found that the presence of rapsyn slows the turnover of AChRs by as much as twofold. The effect was similar for both embryonic (alpha2betadeltagamma) and adult (alpha2betadeltaepsilon) AChRs and for AChRs whose beta subunit lacked a putative tyrosine phosphorylation site. Neither colchicine nor cytochalasin D altered AChR turnover or prevented the rapsyn effect. Mutant rapsyn proteins whose N-terminal myristoylation signal was eliminated, or whose C terminus or zinc-finger domains were deleted, failed to change the rate of receptor turnover. Each of these mutations affects the association of the AChR with rapsyn, suggesting that AChR stability is altered by interaction between the two proteins. Our results suggest that, in addition to its role in AChR clustering, rapsyn also functions to metabolically stabilize the AChR.
To investigate the mechanism of assembly of the mouse muscle acetylcholine receptor, we have expressed truncated N-terminal fragments of the ␣ and ␦ subunits in COS cells and have examined their ability to fold, to associate into heterodimers, and to form a ligand-binding site. Truncated fragments of the ␣ subunit that include all, part, or none of the first transmembrane domain (M1) folded to acquire ␣-bungarotoxin binding activity. Neither the full-length ␣ subunit nor any of the fragments were expressed on the cell surface, although the shortest folded fragment lacking a transmembrane domain was secreted into the medium. When coexpressed with the ␦ subunit, the ␣ subunit fragment possessing M1 formed a heterodimer containing a ligand-binding site, but shorter fragments, which lack transmembrane segments, did not associate with the ␦ subunit. N-terminal ␦ subunit fragments gave similar results. An N-terminal ␦ subunit fragment that contains M1 associated with the ␣ subunit to form a heterodimer, while a fragment lacking M1 did not. These results show that a complete M1 domain is necessary for association of truncated N-terminal ␣ and ␦ subunits into a heterodimer with high affinity ligand binding activity.The nicotinic acetylcholine receptor (AChR) 1 of mammalian muscle and the electrical organ of Torpedo is the best characterized member of a family of ligand-gated ion channels that mediate rapid synaptic transmission in the nervous system (1). All members of the family are thought to have a common pentameric structure in which highly homologous subunits surround a central aqueous pore whose opening and closing is regulated by binding of the neurotransmitter ligand (2-4). The muscle receptor consists of four different subunits in the ratio ␣ 2 ::␥:␦ (5, 6). Each of the subunits is separately synthesized and translocated into the endoplasmic reticulum, where AChR assembly occurs (7-9). Before assembly, the ␣ subunit undergoes a folding reaction whose product can be recognized by its ability to bind ␣-bungarotoxin (␣-BuTx) (10 -12). The other subunits also presumably undergo folding reactions before assembly, although the intermediates have not been characterized. Association of the folded subunits to form the assembled receptor occurs by a defined pathway in which the first step is the formation of the heterodimers ␣␦ and ␣␥ (13-16). The binding of the ␣ subunit to the ␦ and ␥ subunits results in the formation of a ligand-binding site within each heterodimer. Each site is characterized by distinctive properties that correspond to those in the intact AChR (6, 17). Several observations suggest that the binding site for competitive antagonists occurs at or near the interface between ␣ and ␦ subunits or ␣ and ␥ subunits, respectively (18 -20).In previous experiments, we have sought to define the domains of the AChR subunits that participate in the subunit interactions required for heterodimer formation. Chimeric subunits and dominant negative experiments were used to show that the N-terminal domains of the AChR me...
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