Wang Hung-Li, Kuo Yo-Li, Hsu Chia-Yu, Huang Pei-Chen, Li Allen H, Chou An-Hsun, Yeh Tu-Hsueh, Chen Ying-Ling
Department of Physiology, Chang Gung University School of Medicine; Kwei-San, Tao-Yuan, Taiwan, ROC.
Cell Signal. 2006 May;18(5):670-8. doi: 10.1016/j.cellsig.2005.06.009. Epub 2005 Jul 14.
Various cellular signaling pathways induced by nociceptin activation of ORL1 (opioid receptor-like 1 receptor) develop homologous desensitization. Multiple lines of evidence suggest that agonist-induced phosphorylation of serine (Ser)/threonine (Thr) residues at intracellular carboxyl tail leads to homologous desensitization of G protein-coupled receptors. In the present study, we investigated the functional role played by C-terminal Ser/Thr residues in agonist-induced desensitization and phosphorylation of ORL1. In HEK 293 cells expressing wild-type ORL1 and ORL1(CDelta21), which lacks C-terminal 21 amino acids, nociceptin inhibition of adenylate cyclase activity exhibited homologous desensitization after 1 h pretreatment of nociceptin. In contrast, ORL1(CDelta34), which differs with ORL1(CDelta21) by lacking C-terminal Ser(334), Ser(335) and Ser(343) residues, failed to develop agonist-induced desensitization. Point mutant (S343A) ORL1 underwent homologous desensitization after nociceptin pretreatment. Substituting Ser(334) or Ser(335) with alanine greatly impaired nociceptin-induced ORL1 desensitization. In HEK 293 cells expressing double mutant (S334A/S335A) ORL1, nociceptin pretreatment failed to significantly affect the efficacy and potency by which nociceptin inhibits forskolin-stimulated cAMP formation. Mutation of Ser(334) and Ser(335) also greatly reduced nociceptin-induced ORL1 phosphorylation. These results suggest that two C-terminal serine residues, Ser(334) and Ser(335), are required for homologous desensitization and agonist-induced phosphorylation of ORL1.
痛敏肽激活阿片受体样1受体（ORL1）所诱导的多种细胞信号通路会产生同源脱敏。多条证据表明，激动剂诱导的细胞内羧基末端丝氨酸（Ser）/苏氨酸（Thr）残基磷酸化会导致G蛋白偶联受体的同源脱敏。在本研究中，我们调查了C末端Ser/Thr残基在激动剂诱导的ORL1脱敏和磷酸化中所起的功能作用。在表达野生型ORL1和缺少C末端21个氨基酸的ORL1（CDelta21）的HEK 293细胞中，痛敏肽预处理1小时后，痛敏肽对腺苷酸环化酶活性的抑制表现出同源脱敏。相比之下，与ORL1（CDelta21）不同，缺少C末端Ser（334）、Ser（335）和Ser（343）残基的ORL1（CDelta34）未能产生激动剂诱导的脱敏。点突变体（S343A）ORL1在痛敏肽预处理后发生同源脱敏。用丙氨酸取代Ser（334）或Ser（335）极大地损害了痛敏肽诱导的ORL1脱敏。在表达双突变体（S334A/S335A）ORL1的HEK 293细胞中，痛敏肽预处理未能显著影响痛敏肽抑制福斯高林刺激的cAMP形成的效力和效能。Ser（334）和Ser（335）的突变也大大降低了痛敏肽诱导的ORL1磷酸化。这些结果表明，C末端的两个丝氨酸残基Ser（334）和Ser（335）是ORL1同源脱敏和激动剂诱导的磷酸化所必需的。
