Tate B F, Allenby G, Pérez J R, Levin A A, Grippo J F
Department of Metabolic Diseases, Hoffmann-LaRoche, Nutley, New Jersey 07110, USA.
FASEB J. 1996 Nov;10(13):1524-31. doi: 10.1096/fasebj.10.13.8940298.
We previously identified a carboxy-terminal transactivation function termed AF-2 within the last 15 amino acids of the ligand binding domain of the human retinoic acid receptor alpha (hRAR alpha). Truncation of this region abolished transcriptional activity. Here we provide a systematic analysis using alanine scanning mutagenesis of amino acids from Ser405 to Gly419 on a truncated hRAR alpha (delta419) to identify residues within this region that are responsible for transcriptional activity. Whereas mutations in positions 405, 408, 411, and 415-419 have little or no effect on the ability of modified receptors to activate a DR5 response element, mutations in positions 406, 407, 409, 410, and 412-413 modify either the potency or efficacy of all-trans retinoic acid (tRA) -induced gene transcription. Therefore, receptors with mutations in positions 409, 410, 413, and 414 have low transcriptional activity over a wide range of tRA concentrations. Receptors with mutations in positions 406, 407, and 412 exhibit a maximum transcriptional activity similar to wild-type hRAR alpha, but require higher concentrations of tRA. Replacing residues 405-419 on delta419 with the conserved AF-2 domain from the vitamin D3 receptor or the estrogen receptor results in a receptor with wild-type or low transcriptional activity, respectively. A full-length hRAR alpha mutant with an alanine substitution at position 406 (hRAR alpha M406A) binds tRA, but unlike the truncated M406A, which lacks the "F" region, it is not transcriptionally active. Protease mapping experiments detect a consistent difference in the conformation of hRAR alpha M406A compared to wild-type hRAR alpha. These data define amino acids from Ser405 to Gly419 on delta419 that are critical for transcriptional activity and point to the importance of the conformational integrity of receptor domains in maintaining ligand-induced transcriptional activation.
我们之前在人视黄酸受体α（hRARα）配体结合域的最后15个氨基酸内鉴定出一种羧基末端反式激活功能，称为AF-2。该区域的截短消除了转录活性。在此，我们使用丙氨酸扫描诱变对截短的hRARα（δ419）上从Ser405到Gly419的氨基酸进行了系统分析，以确定该区域内负责转录活性的残基。虽然405、408、411和415 - 419位的突变对修饰受体激活DR5反应元件的能力影响很小或没有影响，但406、407、409、410和412 - 413位的突变会改变全反式维甲酸（tRA）诱导的基因转录的效力或效率。因此，409、410、413和414位有突变的受体在广泛的tRA浓度范围内转录活性较低。406、407和412位有突变的受体表现出与野生型hRARα相似的最大转录活性，但需要更高浓度的tRA。用维生素D3受体或雌激素受体的保守AF-2结构域替换δ419上的405 - 419位残基，分别产生具有野生型或低转录活性的受体。在406位有丙氨酸替代的全长hRARα突变体（hRARα M406A）能结合tRA，但与缺乏“F”区域的截短型M406A不同，它没有转录活性。蛋白酶图谱实验检测到hRARα M406A与野生型hRARα相比在构象上存在一致差异。这些数据确定了δ419上从Ser405到Gly419的氨基酸对转录活性至关重要，并指出受体结构域的构象完整性在维持配体诱导的转录激活中的重要性。
