Zhao R, Assaraf Y G, Goldman I D
Departments of Medicine and Molecular Pharmacology, and the Albert Einstein Comprehensive Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Biol Chem. 1998 Jul 24;273(30):19065-71. doi: 10.1074/jbc.273.30.19065.
In an ongoing study of structure-function relationships of the murine reduced folate carrier 1 (RFC1), a glutamate to lysine mutation at amino acid 45 was identified in a methotrexate (MTX)-resistant L1210 clonal variant in which MTX and 5-formyltetrahydrofolate (5-CHO-THF) influx was markedly decreased. The characteristics of the mutated carrier, RFC1-E45K, were studied by cDNA transfection into the murine MTXrA line in which endogenous carrier is not functional. Folic acid influx doubled in the transfectant MTXrA-E45K as compared with L1210 or MTXrA cells; in contrast, MTX and 5-CHO-THF influx was only 14 and 27% that of L1210 cells, respectively. 5-CHO-THF influx in MTXrA-E45K cells was characterized by a 12- and 3.6-fold decrease in influx Vmax and Kt respectively, relative to L1210 cells. The folic acid influx Ki in L1210 cells was more than 50-fold greater than that of MTX based upon inhibition of 5-CHO-THF influx. In comparison, the mutated carrier had comparable affinities for folic acid and MTX in MTXrA-E45K cells due to a 7-fold decrease in the folic acid influx Ki and 7-fold increase in the MTX influx Ki. Transport via native RFC1 is inhibited by a variety of anions in L1210 cells associated with an increase in influx Kt. However, influx of 5-CHO-THF in MTXrA-E45K cells in a HEPES buffer (9 mM chloride) was decreased by 70% due to a 3-fold fall in the Vmax. In the complete absence of chloride (K+-HEPES-sucrose buffer) 5-CHO-THF influx was only 10% that in HBS buffer. 5-CHO-THF influx was restored by addition of chloride, fluoride, or nitrate but not by sulfate, phosphate, or ATP which were all inhibitory over a broad range of concentrations. The data suggest that substitution of a positive for a negative amino acid at position 45 results in the loss of RFC1 mobility in the absence of small inorganic anions that bind to, and neutralize the positive charge on, the lysine residue. Inhibition by higher charged anions may be due to interactions at another carrier site present in both the mutated and wild type carrier. This and other studies suggest that amino acids in the first predicted transmembrane domain play an important role in determining the spectrum of affinities for, and mobility of, RFC1 and is a cluster region for mutations when cells are placed under selective pressure with antifolates that utilize RFC1 as the major route of entry into mammalian cells.
在一项正在进行的关于小鼠还原型叶酸载体1（RFC1）结构-功能关系的研究中，在一种甲氨蝶呤（MTX）耐药的L1210克隆变体中，发现第45位氨基酸发生了谷氨酸到赖氨酸的突变，在该变体中MTX和5-甲酰四氢叶酸（5-CHO-THF）的内流显著减少。通过将cDNA转染到内源性载体无功能的小鼠MTXrA细胞系中，研究了突变载体RFC1-E45K的特性。与L1210或MTXrA细胞相比，转染的MTXrA-E45K细胞中叶酸内流增加了一倍；相比之下，MTX和5-CHO-THF内流分别仅为L1210细胞的14%和27%。与L1210细胞相比，MTXrA-E45K细胞中5-CHO-THF内流的特征是内流Vmax和Kt分别降低了12倍和3.6倍。基于对5-CHO-THF内流的抑制，L1210细胞中叶酸内流的Ki比MTX大50倍以上。相比之下，由于叶酸内流Ki降低了7倍，MTX内流Ki增加了7倍，突变载体在MTXrA-E45K细胞中对叶酸和MTX具有相当的亲和力。在L1210细胞中，通过天然RFC1的转运受到多种阴离子的抑制，这与内流Kt的增加有关。然而，在HEPES缓冲液（9 mM氯离子）中，MTXrA-E45K细胞中5-CHO-THF的内流因Vmax下降3倍而减少了70%。在完全没有氯离子的情况下（K+-HEPES-蔗糖缓冲液），5-CHO-THF内流仅为HBS缓冲液中的10%。添加氯离子、氟离子或硝酸根可恢复5-CHO-THF内流，但添加硫酸根、磷酸根或ATP则不能恢复，这些在广泛的浓度范围内均具有抑制作用。数据表明，在第45位用正氨基酸取代负氨基酸会导致在没有与赖氨酸残基结合并中和其正电荷的小无机阴离子的情况下，RFC1的流动性丧失。带更高电荷的阴离子的抑制作用可能是由于在突变型和野生型载体中都存在的另一个载体位点上的相互作用。这项研究和其他研究表明，第一个预测的跨膜结构域中的氨基酸在确定RFC1的亲和力谱和流动性方面起着重要作用，并且当细胞在以RFC1作为进入哺乳动物细胞的主要途径的抗叶酸药物的选择压力下时，该区域是突变的聚集区。
