title: Role of rapsyn tetratricopeptide repeat and coiled-coil domains in self-association and nicotinic acetylcholine receptor clustering.
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.
authors.affiliation: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: M K
authors.initials: MK
authors.lastname: Ramarao
authors.forename: M J
authors.initials: MJ
authors.lastname: Bianchetta
authors.forename: J
authors.initials: J
authors.lastname: Lanken
authors.forename: J B
authors.initials: JB
authors.lastname: Cohen
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009124:Muscle Proteins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019814:Myristic Acid / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D008164:Luminescent Proteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; C051609:peripheral membrane protein 43K; D019814:Myristic Acid; D049452:Green Fluorescent Proteins; D010749:Phosphoprotein Phosphatases; C089641:protein phosphatase 5