Jones B R, Li W, Cao J, Hoffman T A, Gerk P M, Vore M
Graduate Center for Toxicology, 306 Health Sciences Research Building, University of Kentucky, Lexington, KY 40536-0305, USA.
Mol Pharmacol. 2005 Sep;68(3):701-10. doi: 10.1124/mol.105.013144. Epub 2005 May 25.
Multidrug resistance-associated protein 2 (Mrp2, Abcc2), an organic anion transporter present in the apical membrane of hepatocytes, renal epithelial cells, and enterocytes, is postulated to undergo post-transcriptional regulation. We hypothesized that Mrp2 protein undergoes altered rates of protein synthesis or degradation consistent with different Mrp2 protein expression. We analyzed Mrp2 synthesis, expression, and degradation in control female, 19- and 20-day pregnant, and pregnenolone-16alpha-carbonitrile (PCN)-treated rats using in vivo metabolic-labeling studies with [35S]cysteine/methionine or [14C]NaHCO3, polysomal distribution analyses and ribonuclease protection assays (RPA). Mrp2 protein was significantly increased in rats treated with PCN for 2 days but significantly decreased in 19-day pregnant rats relative to controls; no significant differences were observed in Mrp2 mRNA expression among these groups. The measured half-lives of 14C-labeled Mrp2 in control, pregnant, and PCN-treated rats were 27, 36, and 22 h, respectively, and were not significantly different. The rate of incorporation of 35S into Mrp2 was highest in PCN-treated rats. Polysomal distribution analysis of Mrp2 mRNA was consistent with increased Mrp2 protein synthesis after PCN treatment. The major transcription-initiation site for rat liver determined by RPA was -98 nucleotides (nt), with other start sites observed at -213, -163, -132, and -71 nt; use of transcription sites did not differ among the groups. Differences in the degradation of Mrp2 protein cannot explain the post-transcriptional regulation of Mrp2 in control, pregnant, and PCN-treated rats. Rather, the observed difference in protein synthesis suggests an intrinsic role for the translational regulation of rat Mrp2 protein.
多药耐药相关蛋白2（Mrp2，Abcc2）是一种存在于肝细胞、肾上皮细胞和肠上皮细胞顶膜的有机阴离子转运体，推测其受到转录后调控。我们假设Mrp2蛋白的合成或降解速率发生改变，这与不同的Mrp2蛋白表达情况一致。我们使用[35S]半胱氨酸/甲硫氨酸或[14C]NaHCO3进行体内代谢标记研究、多核糖体分布分析和核糖核酸酶保护试验（RPA），分析了对照雌性大鼠、妊娠19天和20天的大鼠以及孕烯醇酮-16α-腈（PCN）处理的大鼠中Mrp2的合成、表达和降解情况。相对于对照组，PCN处理2天的大鼠中Mrp2蛋白显著增加，但妊娠19天的大鼠中Mrp2蛋白显著降低；这些组之间Mrp2 mRNA表达未观察到显著差异。在对照、妊娠和PCN处理的大鼠中，14C标记的Mrp2的测量半衰期分别为27、36和22小时，且无显著差异。PCN处理的大鼠中35S掺入Mrp2的速率最高。Mrp2 mRNA的多核糖体分布分析与PCN处理后Mrp2蛋白合成增加一致。通过RPA确定的大鼠肝脏主要转录起始位点为-98个核苷酸（nt），在-213、-163、-132和-71 nt处观察到其他起始位点；各组之间转录位点的使用情况没有差异。Mrp2蛋白降解的差异不能解释对照、妊娠和PCN处理的大鼠中Mrp2的转录后调控。相反，观察到的蛋白合成差异表明大鼠Mrp2蛋白的翻译调控具有内在作用。
