Mechanism of nicotinic acetylcholine receptor cluster formation by rapsyn

Rapsyn, a peripheral membrane protein of skeletal muscle, clusters nicotinic acetylcholine receptors (nAChRs) at high density in the postsynaptic membrane, The mechanism of nAChR clustering by rapsyn was analyzed by expressing nAChRs in HEK293T cells with various fragments of mouse rapsyn fused to green fluorescent protein, Membrane targeting of rapsyn is conferred solely by its acylated N terminus, as the myristoylated N-terminal 15 amino acids of rapsyn are sufficient to target green fluorescent protein to the plasma membrane. However, neither N-terminal myristoylation nor the conserved N-terminal amino acid sequence is essential, Membrane targeting, self-association, and nAChR clustering are preserved when the first 10 amino acids of rapsyn were replaced by those of src, which also contains a consensus sequence for N-myristoylation, or by those of GAP43, which contains a palmitoylation sequence, Rapsyn(1-90), containing two tetratrichopeptide repeats is sufficient for self-association. Rapsyn(1-360), lacking the cysteine rich domain, clusters nAChRs, while rapsyn(1-287), containing seven tetratrichopeptide repeats, does not cluster nAChRs, We identified rapsyn(298-331) as a potential coiled-coil domain, and established that mutations disrupting coiled-coil propensity prevent nAChR clustering, Thus the structural domains of rapsyn necessary for membrane targeting, self-association: and nAChR clustering are distinct, with nAChR-rapsyn interaction mediated by a previously unrecognized coiled-coil motif.