Abstract
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 NH(2)-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-(black triangle288-348)), 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-H2 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.
Highlights
A characteristic and important feature of the vertebrate neuromuscular junction is the clustering of nicotinic acetylcholine receptors[1] at high surface density (ϳ10,000/2) in the post-synaptic membrane underlying the nerve terminal, with the nAChR surface density decreasing by a factor of 100 –1000 within a few microns
The results presented here extend our understanding of the role of the rapsyn coiled-coil and tetratricopeptide repeats (TPRs) domains in nAChR clustering and self-association, respectively
Internal deletion of the rapsyn sequence corresponding to the coiled-coil domain (rapsyn-(Œ288 –348)) completely abolished nAChR clustering without affecting rapsyn self-association
Summary
A characteristic and important feature of the vertebrate neuromuscular junction is the clustering of nicotinic acetylcholine receptors (nAChR)[1] at high surface density (ϳ10,000/2) in the post-synaptic membrane underlying the nerve terminal, with the nAChR surface density decreasing by a factor of 100 –1000 within a few microns. Rapsyn-(Œ288 –348)-GFP—This construct encodes full-length rapsyn with a deletion of amino acids 288 –348 which contain the coiled-coil domain (6). In experiments involving rapsyn mutants with TPR deletions, in some cells nAChRs were seen both clustered with rapsyn and distributed diffusely on the surface.
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