Homologous desensitization of human corticotropin-releasing factor(1) receptor in stable transfected mouse fibroblast cells
Homologous desensitization of human corticotropin-releasing factor(1) receptor in stable transfected mouse fibroblast cells
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DOI：
10.1016/0006-8993(95)01480-2
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发表时间：
1996-02-26
期刊：
影响因子：
2.9
通讯作者：
DeSouza, EB
中科院分区：
文献类型：
--
作者：
Dieterich, KD;Grigoriadis, DE;DeSouza, EB
先前的放射性配体结合和第二信使研究表明，促肾上腺皮质激素释放因子（CRF）在体内和体外治疗后均可调节其受体。在本研究中，我们确定了导致稳定转染CRF(1) DNA（来自人垂体）的小鼠成纤维细胞（Ltk(-)）中克隆CRF受体的CRF诱导下调和脱敏的事件顺序。用大鼠/人CRF处理细胞产生[I-125]Tyr(0)-绵羊CRF ([I-125]oCRF)结合的剂量和时间依赖性减少，并伴随CRF刺激的腺苷酸环化酶活性的减少。 CRF 治疗几分钟后，[I-125]oCRF 结合和激动剂刺激的 cAMP 产生显着减少，CRF 治疗 1 小时后观察到最大减少（60-80%）。 Scatchard 分析表明，[I-125]oCRF 结合的减少是由于受体的下调，而配体的亲和力没有明显改变。由于转染细胞系是使用人工启动子设计的，因此在 CRF 处理长达 24 小时后，我们没有检测到 CRF(1) 受体 mRNA 水平有任何显着变化。
Previous radioligand binding and second messenger studies have shown that corticotropin-releasing factor (CRF) modulates its receptor following both in vivo and in vitro treatment. In the present study, we determined the sequence of events leading to CRF-induced downregulation and desensitization of cloned CRF receptors in murine fibroblast cells (Ltk(-)) stably transfected with CRF(1) DNA (from human pituitary). Treatment of cells with rat/human CRF produced a dose- and time-dependent decrease in [I-125]Tyr(0)-ovine CRF ([I-125]oCRF) binding and a concomitant decrease in CRF-stimulated adenylate cyclase activity. Significant decreases in [I-125]oCRF binding and agonist-stimulated cAMP production were evident minutes after CRF treatment with maximal (60-80%) reductions seen following 1 h of CRF treatment. Scatchard analysis revealed that the decrease in [I-125]oCRF binding was due to the downregulation of the receptor with no significant alteration seen in the affinity of the ligand. Since the transfected cell line is engineered using an artificial promoter, we did not detect any significant changes in CRF(1) receptor mRNA levels following CRF treatment for up to 24 h.