A role for the calmodulin kinase II-related anchoring protein (αkap) in maintaining the stability of nicotinic acetylcholine receptors.
A role for the calmodulin kinase II-related anchoring protein (αkap) in maintaining the stability of nicotinic acetylcholine receptors.
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DOI：
10.1523/jneurosci.6477-11.2012
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发表时间：
2012-04-11
期刊：
影响因子：
--
通讯作者：
Akaaboune M
中科院分区：
文献类型：
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作者：
Mouslim C;Aittaleb M;Hume RI;Akaaboune M
αkap是Camk 2a基因编码的肌肉特异性锚定蛋白，被认为在将多种钙/钙调蛋白激酶II亚型靶向特定亚细胞位置中发挥作用。在这里，我们证明了αkap在稳定烟碱乙酰胆碱受体（AChR）的新功能。αkap表达的shRNA敲低显著增强了AChR α亚基（AChRα）的降解，导致分化的C2 C12肌管表面AChR簇更少更小。HEK 293 T细胞的突变和生化研究表明，αkap通过泛素依赖性机制促进AChRα的稳定性。在缺乏αkap的情况下，AChRα被大量泛素化，蛋白酶体抑制剂可增加AChRα的数量。然而，在αkap存在的情况下，AChRα的泛素化程度较低，蛋白酶体抑制剂对AChRα的积累几乎没有影响。AChRα泛素化的主要位点位于大的细胞内环内，在αkap缺失的情况下，该环中关键的赖氨酸残基突变为精氨酸增加了AChRα的稳定性。这些结果提供了一种意想不到的机制，通过这种机制αkap控制受体运输到肌细胞表面，从而维持突触后受体密度和突触功能。
αkap, a muscle specific anchoring protein encoded within the Camk2a gene is thought to play a role in targeting multiple calcium/calmodulin kinase II isoforms to specific subcellular locations. Here we demonstrate a novel function of αkap in stabilizing nicotinic acetylcholine receptors (AChR). Knockdown of αkap expression with shRNA significantly enhanced the degradation of AChR α-subunits (AChRα), leading to fewer and smaller AChR clusters on the surface of differentiated C2C12 myotubes. Mutagenesis and biochemical studies in HEK293T cells revealed that αkap promoted AChRα stability by a ubiquitin-dependent mechanism. In the absence of αkap, AChRα was heavily ubiquitinated and the number of AChRα was increased by proteasome inhibitors. However, in the presence of αkap, AChRα was less ubiquitinated and proteasome inhibitors had almost no effect on AChRα accumulation. The major sites of AChRα ubiquitination reside within the large intracellular loop and mutations of critical lysine residues in this loop to arginine increased AChRα stability in the absence of αkap. These results provide an unexpected mechanism by which αkap controls receptor trafficking onto the surface of muscle cells, and thus the maintenance of postsynaptic receptor density and synaptic function.