Role of rapsyn tetratricopeptide repeat and coiled-coil domains in self-association and nicotinic acetylcholine receptor clustering

Rapsyn, a 43-kDa peripheral membrane protein of skeletal muscle, is essential for clustering nicotinic acetylcholine receptors (nAChR) in the postsynaptic membrane, Previous studies with rapsyn NH2-terminal fragments fused to green fluorescent protein, expressed in 293T cells along with nAChRs, establish the following: Rapsyn-(1-90), containing the myristoylated amino terminus and two tetratricopeptide repeats (TPRs), was sufficient for self-association at the plasma membrane; rapsyn-(1-287), containing seven TPRs, did not cluster nAChRs; whereas rapsyn-(1-360), containing a coiled-coil domain (rapsyn-(298-331)), clustered nAChRs, To further analyze the role of rapsyn structural domains in self-association and nAChR clustering, we have characterized the clustering properties of additional rapsyn mutants containing deletions and substitutions within the TPR and coiled-coil domains. A mutant lacking the coiled-coil domain alone (rapsyn-(Delta 288-348)), failed to cluster nAChRs, Within the coiled-coil domain neutralization of the charged side chains was tolerated, while alanine substitutions of large hydrophobic residues resulted in the loss of nAChR clustering. Rapsyn self-association requires at least two TPRs, as a single TPR (TPR1 or TPR2 alone) was not sufficient. While TPRs 1 and 2 are sufficient for self-association, they are not necessary, as TPRs 3-7 also formed clusters similar to wild-type rapsyn, Fragments containing TPRs co-localized with full-length rapsyn, while the expressed coiled-coil or RING-MB domain did not, These results are discussed in terms of a homology model of rapsyn, based on the three-dimensional structure of the TPR domain of protein phosphatase 5.