Carboxyl-terminal splicing enhances physical interactions between the cytoplasmic tails of purinergic P2X receptors
Carboxyl-terminal splicing enhances physical interactions between the cytoplasmic tails of purinergic P2X receptors
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DOI：
10.1124/mol.105.019802
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发表时间：
2006-05-01
影响因子：
3.6
通讯作者：
Tsujimoto, G
中科院分区：
文献类型：
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作者：
Koshimizu, T;Kretschmannova, K;Tsujimoto, G
嘌呤能 P2X 受体是由三个亚基组成的离子传导通道，每个亚基具有两个跨膜结构域和细胞内氨基 (N) 和羧基 (C) 末端。尽管选择性剪接广泛地修饰了 P2X 亚基的 C 端序列，但这种转录后修饰对受体结构和功能的直接影响仍然知之甚少。在这项研究中，我们重点关注小鼠垂体 P2X(2) 受体。在该组织中，P2X(2a) C 末端的渐进剪接产生了两个功能亚基变体：P2X(2b) 和 P2X(2e)，当受体处于同聚状态时，它们表现出加速的脱敏率和减弱的钙信号。为了测量活细胞中亚基间的相互作用，使用了荧光素酶和附着在这些亚基的 N 或 C 亚基末端的荧光蛋白之间生物发光共振能量的有效转移。与阴性对照相比，荧光/发光强度比显着增加，检测到 P2X2a 受体全长 C 末端之间的组成型相互作用。此外，在含有P2X(2b)或P2X(2e)亚基的同聚和异聚受体中，C末端之间以及相邻亚基的C末端和N末端之间的相互作用显着增强。最后，在剪接连接处删除两个氨基酸，但不在P2X(2b)受体的C末端，导致通道脱敏和发光共振能量转移的增强。这些结果表明，C 端结构在细胞质亚基间相互作用中起着关键作用，并表明在 ATP 应用之前亚基相互作用的程度可能有助于随后与激动剂依赖性脱敏相关的通道活性和构象变化。
Purinergic P2X receptors are ion-conducting channels composed of three subunits, each having two transmembrane domains and intracellular amino (N) and carboxyl (C) termini. Although alternative splicing extensively modifies the C-terminal sequences of P2X subunits, the direct influence of such post-transcriptional modifications on receptor architecture and function remains poorly understood. In this study, we focused on mouse pituitary P2X(2) receptors. In this tissue, progressive splicing of the P2X(2a) C terminus generated two functional subunit variants, P2X(2b) and P2X(2e), which exhibited accelerated desensitization rates and attenuated calcium signals when the receptors were in homomeric states. To measure the intersubunit interaction in living cells, the efficient transfer of bioluminescent resonance energy between luciferase and fluorescent proteins attached to the N- or C-subunit termini of these subunits was used. The constitutive interactions between the full-length C termini of P2X2a receptor were detected by a significant increase in fluorescence/luminescence intensity ratio compared with negative controls. Moreover, interactions between C termini and between C- and N termini of adjacent subunits were significantly enhanced in homomeric and heteromeric receptors containing P2X(2b) or P2X(2e) subunits. Finally, deletion of two amino acids at the splicing junction, but not at the C-terminal end of the P2X(2b) receptor, resulted in the enhancement of channel desensitization and luminescence resonance energy transfer. These results indicate that C-terminal structure plays a critical role in the cytoplasmic intersubunit interactions and suggest that the extent of subunit interactions before ATP application could contribute to the subsequent channel activity and conformation changes associated with agonist-dependent desensitization.