Analysis of the C-terminal tail of the rat thyrotropin-releasing hormone receptor-1 in interactions and cointernalization with β-arrestin 1-green fluorescent protein
Analysis of the C-terminal tail of the rat thyrotropin-releasing hormone receptor-1 in interactions and cointernalization with β-arrestin 1-green fluorescent protein
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DOI：
10.1124/mol.59.2.375
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发表时间：
2001-02-01
影响因子：
3.6
通讯作者：
Milligan, G
中科院分区：
文献类型：
--
作者：
Groarke, DA;Drmota, T;Milligan, G
在人胚肾293细胞中共表达大鼠促甲状腺激素释放激素受体-1与β-抑制蛋白1-绿色荧光蛋白（GFP）导致抑制蛋白激动剂依赖性移位至质膜，随后与受体共内化。受体C-末端尾从93至50个氨基酸的截短没有改变这一点。截短到少于47个氨基酸阻止了这种相互作用，并抑制但不能完全消除激动剂诱导的受体内化。C-末端尾部的缺失和定点突变体表明，单独消除潜在的酪蛋白激酶II磷酸化位点或网格蛋白/网格蛋白衔接子基序不足以阻止受体的内化或其与β-抑制蛋白1-GFP的共内化。酰化位点的改变减少了内化并阻止了与β-抑制蛋白1-GFP的相互作用。这些突变体的组合导致缺乏与β-抑制蛋白1-GFP的相互作用和受体内化的10倍减少。尽管如此，缺乏三个蛋白质序列基序的受体构建体是完全功能性的。这些研究绘制了促进促甲状腺激素释放激素受体-1C-末端尾与β-抑制蛋白1-GFP有效接触所需的相互作用的位点，并表明这些相互作用在受体的激动剂诱导的内化中的关键作用。
Coexpression of the rat thyrotropin releasing hormone receptor-1 with beta -arrestin 1-green fluorescent protein (GFP) in human embryonic kidney 293 cells results in agonist-dependent translocation of the arrestin to the plasma membrane followed by its cointernalization with the receptor. Truncations of the receptor C-terminal tail from 93 to 50 amino acids did not alter this. Truncations to fewer than 47 amino acids prevented such interactions and inhibited but did not fully eliminate agonist-induced internalization of the receptor. Deletion and site-directed mutants of the C-terminal tail indicated that separate elimination of a potential casein kinase II phosphorylation site or clathrin/clathrin adapter motifs was insufficient to prevent either internalization of the receptor or its cointernalization with beta -arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with beta -arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with beta -arrestin 1-GFP and a 10-fold reduction in internalization of the receptor. Despite this, the receptor construct that lacked the three protein sequence motifs was fully functional. These studies map sites that contribute the interactions of the thyrotropin releasing hormone receptor-1 C-terminal tail required for effective contacts with beta -arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the receptor.