Stewart Richard, Zissimopoulos Spyros, Lai F Anthony
Wales Heart Research Institute, Department of Cardiology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK.
Biochem J. 2003 Dec 15;376(Pt 3):795-9. doi: 10.1042/BJ20030597.
The C-terminal 100 amino acids of the RyR (ryanodine receptor), referred to as the C-terminal tail, is a highly conserved sequence that is present in all known RyR isoforms and which has been implicated in channel function. Deleting the final 15 amino acids from the full-length skeletal muscle RyR resulted in an inactive channel, attributed to impaired assembly of a tetrameric RyR complex [Gao, Tripathy, Lu and Meissner (1997) FEBS Lett. 412, 223-226]. To account for these observations, the C-terminal tail itself may be an important molecular determinant of oligomerization. Alternatively, the large N-terminal cytoplasmic domain may fold back upon itself to interact with the C-terminal tail to provide a correctly folded tetrameric structure. We explored these possibilities for RyR2 (cardiac RyR) using the yeast two-hybrid interaction assay and in vitro translation followed by immunoprecipitation and chemical cross-linking. The data indicate that the C-terminal tail of RyR2 is capable of self-tetramerization. Moreover, a truncated C-terminal tail, lacking the final 15 amino acids, failed to self-associate. These observations suggest that the intrinsic ability of the RyR C-terminal tail to self-tetramerize may be vitally important for the oligomeric assembly of the native RyR channel.
兰尼碱受体(RyR)的C末端100个氨基酸,被称为C末端尾巴,是一个高度保守的序列,存在于所有已知的RyR亚型中,并且与通道功能有关。从全长骨骼肌RyR中删除最后15个氨基酸会导致通道失活,这归因于四聚体RyR复合物组装受损[高、特里帕蒂、卢和迈斯纳(1997年)《欧洲生物化学学会联合会快报》412卷,223 - 226页]。为了解释这些观察结果,C末端尾巴本身可能是寡聚化的重要分子决定因素。或者,大的N末端胞质结构域可能自身折叠回来与C末端尾巴相互作用,以提供正确折叠的四聚体结构。我们使用酵母双杂交相互作用测定法以及体外翻译,随后进行免疫沉淀和化学交联,探索了RyR2(心脏RyR)的这些可能性。数据表明,RyR2的C末端尾巴能够自我四聚化。此外,缺少最后15个氨基酸的截短C末端尾巴无法自我缔合。这些观察结果表明,RyR C末端尾巴自我四聚化的内在能力对于天然RyR通道的寡聚体组装可能至关重要。