Dieterich K D, Grigoriadis D E, De Souza E B
Neurocrine Biosciences, Inc., San Diego, CA 92121, USA.
Brain Res. 1996 Feb 26;710(1-2):287-92. doi: 10.1016/0006-8993(95)01480-2.
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 CRF1 DNA (from human pituitary). Treatment of cells with rat/human CRF produced a dose- and time-dependent decrease in [125I]Tyr degrees-ovine CRF ([125I]oCRF) binding and a concomitant decrease in CRF-stimulated adenylate cyclase activity. Significant decreases in [125I]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 [125I]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 CRF1 receptor mRNA levels following CRF treatment for up to 24 h.
以往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）在体内和体外处理后均会调节其受体。在本研究中，我们确定了在用CRF1 DNA（来自人垂体）稳定转染的鼠成纤维细胞（Ltk-）中，导致CRF诱导的克隆CRF受体下调和脱敏的事件顺序。用大鼠/人CRF处理细胞导致[125I]酪氨酸化羊CRF（[125I]oCRF）结合呈剂量和时间依赖性降低，同时CRF刺激的腺苷酸环化酶活性也降低。在用CRF处理数分钟后，[125I]oCRF结合和激动剂刺激的cAMP产生显著降低，在CRF处理1小时后出现最大（60-80%）降低。Scatchard分析表明，[125I]oCRF结合的降低是由于受体下调，而配体亲和力未见明显改变。由于转染细胞系是使用人工启动子构建的，在CRF处理长达24小时后未检测到CRF1受体mRNA水平有任何显著变化。
