Hanyaloglu A C, Vrecl M, Kroeger K M, Miles L E, Qian H, Thomas W G, Eidne K A
7TM Receptor Laboratory, Western Australian Institute for Medical Research, Keogh Institute for Medical Research, Sir Charles Gairdner Hospital, and Animal Sciences, University of Western Australia, Perth, Western Australia 6009, Australia.
J Biol Chem. 2001 May 25;276(21):18066-74. doi: 10.1074/jbc.M009275200. Epub 2001 Mar 9.
We have previously shown that the mammalian gonadotropin-releasing hormone receptor (GnRHR), a unique G-protein-coupled receptor (GPCR) lacking an intracellular carboxyl tail (C-tail), does not follow a beta-arrestin-dependent internalization pathway. However, internalization of a chimeric GnRHR with the thyrotropin-releasing hormone receptor (TRHR) C-tail does utilize beta-arrestin. Here, we have investigated the sites within the intracellular C-tail domain that are important for conferring beta-arrestin-dependent internalization. In contrast to the chimeric GnRHR with a TRHR C-tail, a chimeric GnRHR with the catfish GnRHR C-tail is not beta-arrestin-dependent. Sequence comparisons between these chimeric receptors show three consensus phosphorylation sites for casein kinase II (CKII) in the TRHR C-tail but none in the catfish GnRHR C-tail. We thus investigated a role for CKII sites in determining GPCR internalization via beta-arrestin. Sequential introduction of three CKII sites into the chimera with the catfish C-tail (H354D,A366E,G371D) resulted in a change in the pattern of receptor phosphorylation and beta-arrestin-dependence, which only occurred when all three sites were introduced. Conversely, mutation of the putative CKII sites (T365A,T371A,S383A) in the C-tail of a beta-arrestin-sensitive GPCR, the TRHR, resulted in decreased receptor phosphorylation and a loss of beta-arrestin-dependence. Mutation of all three CKII sites was necessary before a loss of beta-arrestin-dependence was observed. Visualization of beta-arrestin/GFP redistribution confirmed a loss or gain of beta-arrestin sensitivity for receptor mutants. Internalization of receptors without C-tail CKII sites was promoted by a phosphorylation-independent beta-arrestin mutant (R169E), suggesting that these receptors do not contain the necessary phosphorylation sites required for beta-arrestin-dependent internalization. Apigenin, a specific CKII inhibitor, blocked the increase in receptor internalization by beta-arrestin, thus providing further support for the involvement of CKII. This study presents evidence of a novel role for C-tail CKII consensus sites in targeting these GPCRs to the beta-arrestin-dependent pathway.
我们之前已经表明，哺乳动物促性腺激素释放激素受体（GnRHR）是一种独特的G蛋白偶联受体（GPCR），缺乏细胞内羧基末端（C末端），不遵循β-抑制蛋白依赖性内化途径。然而，带有促甲状腺激素释放激素受体（TRHR）C末端的嵌合GnRHR的内化确实利用了β-抑制蛋白。在这里，我们研究了细胞内C末端结构域中对于赋予β-抑制蛋白依赖性内化很重要的位点。与带有TRHR C末端的嵌合GnRHR不同，带有鲶鱼GnRHR C末端的嵌合GnRHR不是β-抑制蛋白依赖性的。这些嵌合受体之间的序列比较显示，TRHR C末端有三个酪蛋白激酶II（CKII）的共有磷酸化位点，而鲶鱼GnRHR C末端没有。因此，我们研究了CKII位点在通过β-抑制蛋白决定GPCR内化中的作用。将三个CKII位点依次引入带有鲶鱼C末端的嵌合体（H354D、A366E、G371D）导致受体磷酸化模式和β-抑制蛋白依赖性发生变化，这种变化只有在引入所有三个位点时才会出现。相反，β-抑制蛋白敏感的GPCR即TRHR的C末端中假定的CKII位点（T365A、T371A、S383A）发生突变，导致受体磷酸化减少以及β-抑制蛋白依赖性丧失。在观察到β-抑制蛋白依赖性丧失之前，所有三个CKII位点都发生突变是必要的。β-抑制蛋白/绿色荧光蛋白（GFP）重新分布的可视化证实了受体突变体对β-抑制蛋白敏感性的丧失或增加。没有C末端CKII位点的受体的内化由磷酸化非依赖性的β-抑制蛋白突变体（R169E）促进，这表明这些受体不包含β-抑制蛋白依赖性内化所需的必要磷酸化位点。芹菜素是一种特异性CKII抑制剂，它阻断了β-抑制蛋白引起的受体内化增加，从而为CKII的参与提供了进一步的支持。这项研究提供了证据，证明C末端CKII共有位点在将这些GPCR靶向β-抑制蛋白依赖性途径中具有新的作用。
