Ramarao M K, Bianchetta M J, Lanken J, Cohen J B
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2001 Mar 9;276(10):7475-83. doi: 10.1074/jbc.M009888200. Epub 2000 Nov 21.
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.
Rapsyn是一种43 kDa的骨骼肌外周膜蛋白，对于烟碱型乙酰胆碱受体（nAChR）在突触后膜的聚集至关重要。先前将rapsyn氨基末端片段与绿色荧光蛋白融合的研究，与nAChRs一起在293T细胞中表达，得出以下结果：包含肉豆蔻酰化氨基末端和两个四肽重复序列（TPR）的Rapsyn-(1-90)足以在质膜上进行自我结合；包含七个TPR的rapsyn-(1-287)不能使nAChRs聚集；而包含卷曲螺旋结构域（rapsyn-(298-331)）的rapsyn-(1-360)使nAChRs聚集。为了进一步分析rapsyn结构域在自我结合和nAChR聚集中的作用，我们对在TPR和卷曲螺旋结构域内包含缺失和替换的其他rapsyn突变体的聚集特性进行了表征。仅缺少卷曲螺旋结构域的突变体（rapsyn-(black triangle288-348)）不能使nAChRs聚集。在卷曲螺旋结构域内，带电侧链的中和是可以耐受的，而大的疏水残基被丙氨酸取代会导致nAChR聚集的丧失。Rapsyn自我结合至少需要两个TPR，因为单个TPR（单独的TPR1或TPR2）是不够的。虽然TPR1和TPR2足以进行自我结合，但它们不是必需的，因为TPR3-7也形成了与野生型rapsyn相似的聚集体。包含TPR的片段与全长rapsyn共定位，而表达的卷曲螺旋或RING-H2结构域则没有。根据rapsyn的同源模型，基于蛋白磷酸酶5的TPR结构域的三维结构对这些结果进行了讨论。
