Regulation of corticotropin-releasing hormone receptor type 1alpha signaling: structural determinants for G protein-coupled receptor kinase-mediated phosphorylation and agonist-mediated desensitization.
Regulation of corticotropin-releasing hormone receptor type 1alpha signaling: structural determinants for G protein-coupled receptor kinase-mediated phosphorylation and agonist-mediated desensitization.
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DOI：
10.1210/me.2004-0275
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发表时间：
2005-02
影响因子：
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通讯作者：
T. Teli;D. Markovic;M. Levine;E. Hillhouse;D. Grammatopoulos
中科院分区：
文献类型：
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作者：
T. Teli;D. Markovic;M. Levine;E. Hillhouse;D. Grammatopoulos
CRH受体1型（CRH-R1）信号传导活性的减弱可能涉及CRH-R1与细胞内效应物的脱敏和解偶联。我们研究了天然CRH-R在来自孕妇的人子宫肌层细胞中的脱敏作用以及在人胚肾（HEK）293细胞中稳定过表达的重组CRH-R1 α。在这两种细胞类型中，CRH-R1介导的腺苷酸环化酶激活对高浓度CRH预处理诱导的同源脱敏敏感。时程研究显示，预处理约40 min后出现半数最大脱敏，CRH-R1 α功能反应在去除CRH预处理后2 h内完全恢复。在HEK 293细胞中，CRH-R1 α的脱敏与受体磷酸化和随后的内吞作用有关。为了分析导致CRH-R1 α脱敏的机制，我们过表达截短的β-抑制蛋白（319-418），并进行免疫共沉淀和G蛋白偶联受体激酶（GRK）易位研究。我们发现GRK 3和GRK 6是与CRH-R1 α相互作用的主要亚型，GRK 3的募集需要G β γ亚基和β抑制蛋白。通过对CRH-R1 α C端的Ser和Thr残基进行定点突变，发现Thr 399对GRK诱导的受体磷酸化和脱敏起重要作用，我们得出结论，CRH-R1 α的同源脱敏涉及多种GRK亚型、G β γ二聚体和β-arrestin的协同作用。基于我们对GRK依赖性磷酸化的关键氨基酸的鉴定，我们证明了CRH-R1 α羧基尾对受体活性调节的重要性。
Attenuation of CRH receptor type 1 (CRH-R1) signaling activity might involve desensitization and uncoupling of CRH-R1 from intracellular effectors. We investigated the desensitization of native CRH-R in human myometrial cells from pregnant women and recombinant CRH-R1alpha stably overexpressed in human embryonic kidney (HEK) 293 cells. In both cell types, CRH-R1-mediated adenylyl cyclase activation was susceptible to homologous desensitization induced by pretreatment with high concentrations of CRH. Time course studies showed half-maximal desensitization occurring after approximately 40 min of pretreatment and full recovery of CRH-R1alpha functional response within 2 h of removal of CRH pretreatment. In HEK 293 cells, desensitization of CRH-R1alpha was associated with receptor phosphorylation and subsequent endocytosis. To analyze the mechanism leading to CRH-R1alpha desensitization, we overexpressed a truncated beta-arrestin (319-418) and performed coimmunoprecipitation and G protein-coupled receptor kinase (GRK) translocation studies. We found that GRK3 and GRK6 are the main isoforms that interact with CRH-R1alpha, and that recruitment of GRK3 requires Gbetagamma-subunits as well as beta-arrestin. Site-directed mutagenesis of Ser and Thr residues in the CRH-R1alpha C terminus, identified Thr399 as important for GRK-induced receptor phosphorylation and desensitization.We conclude that homologous desensitization of CRH-R1alpha involves the coordinated action of multiple GRK isoforms, Gbeta gamma dimers and beta-arrestin. Based on our identification of key amino acid(s) for GRK-dependent phosphorylation, we demonstrate the importance of the CRH-R1alpha carboxyl tail for regulation of receptor activity.