Carboxyl-terminal and intracellular loop sites for CRF1 receptor phosphorylation and beta-arrestin-2 recruitment: a mechanism regulating stress and anxiety responses.
Carboxyl-terminal and intracellular loop sites for CRF1 receptor phosphorylation and beta-arrestin-2 recruitment: a mechanism regulating stress and anxiety responses.
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CRF1 受体磷酸化和 β-arrestin-2 募集的羧基末端和细胞内环位点：调节压力和焦虑反应的机制。
DOI：
10.1152/ajpregu.00099.2006
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Hauger,RichardL
中科院分区：
文献类型：
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作者：
Oakley,RobertH;Olivares-Reyes,JAlberto;Hudson,ChristineC;Flores-Vega,Fabiola;Dautzenberg,FrankM;Hauger,RichardL
主要目的是验证激动剂诱导的促肾上腺皮质激素释放因子1型(CRF1)受体磷酸化是β-arrestins从胞浆转移到细胞膜的假设。我们还试图确定CRF1受体羧基末端和第三细胞内环中基序对β-arrestin募集的相对重要性。在多种细胞系中，β-arrestin-2比β-arrestin-1更快地转运到激动剂激活的膜CRF1受体上。尽管CRF1受体被激动剂处理内化，但两种arrestin亚型都没有与受体在细胞内运输，这表明CRF1受体-arrestin复合体在细胞膜或细胞膜附近解离。CRF1受体的内化涉及arrestin和clathrin两种依赖机制。为了研究介导β-arrestin-2-CRF1受体相互作用的分子决定因素，我们进行了突变以去除潜在的G蛋白偶联受体激酶磷酸化位点。在丝氨酸-386位截断CRF1受体的羧基末端极大地减少了激动剂依赖的磷酸化，但仅部分地损害了β-arrestin-2的募集。去除细胞内第三环中的丝氨酸/苏氨酸簇也显著降低CRF1受体的磷酸化，但不改变β-arrestin-2的募集。同时具有这两种突变的CRF1受体的磷酸化被取消。令人惊讶的是，这个突变体仍然招募了β-arrestin-2。这些突变不改变CRF1受体的膜表达或cAMP信号。我们的数据揭示了至少以下两个不同的受体区域参与β-arrestin-2的募集：1)其中丝氨酸/苏氨酸残基必须被磷酸化的羧基末端基序和2)由激动剂诱导的CRF1受体构象变化而配置的细胞内环状基序。β-arrestin-2-CRF1受体相互作用不足可能通过诱导过量的CRF1受体信号参与情感性精神障碍的病理生理过程。
The primary goal was to test the hypothesis that agonist-induced corticotropin-releasing factor type 1 (CRF1) receptor phosphorylation is required for β-arrestins to translocate from cytosol to the cell membrane. We also sought to determine the relative importance to β-arrestin recruitment of motifs in the CRF1receptor carboxyl terminus and third intracellular loop. β-Arrestin-2 translocated significantly more rapidly than β-arrestin-1 to agonist-activated membrane CRF1receptors in multiple cell lines. Although CRF1receptors internalized with agonist treatment, neither arrestin isoform trafficked with the receptor inside the cell, indicating that CRF1receptor-arrestin complexes dissociate at or near the cell membrane. Both arrestin and clathrin-dependent mechanisms were involved in CRF1receptor internalization. To investigate molecular determinants mediating the robust β-arrestin-2-CRF1receptor interaction, mutagenesis was performed to remove potential G protein-coupled receptor kinase phosphorylation sites. Truncating the CRF1receptor carboxyl terminus at serine-386 greatly reduced agonist-dependent phosphorylation but only partially impaired β-arrestin-2 recruitment. Removal of a serine/threonine cluster in the third intracellular loop also significantly reduced CRF1receptor phosphorylation but did not alter β-arrestin-2 recruitment. Phosphorylation was abolished in a CRF1receptor possessing both mutations. Surprisingly, this mutant still recruited β-arrestin-2. These mutations did not alter membrane expression or cAMP signaling of CRF1receptors. Our data reveal the involvement of at least the following two distinct receptor regions in β-arrestin-2 recruitment:1) a carboxyl-terminal motif in which serine/threonine residues must be phosphorylated and2) an intracellular loop motif configured by agonist-induced changes in CRF1receptor conformation. Deficient β-arrestin-2-CRF1receptor interactions could contribute to the pathophysiology of affective disorders by inducing excessive CRF1receptor signaling.