MDR1 synonymous polymorphisms alter transporter specificity and protein stability in a stable epithelial monolayer.
MDR1 synonymous polymorphisms alter transporter specificity and protein stability in a stable epithelial monolayer.
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DOI：
10.1158/0008-5472.can-13-2064
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发表时间：
2014-01-15
期刊：
影响因子：
11.2
通讯作者：
Gottesman MM
中科院分区：
文献类型：
--
作者：
Fung KL;Pan J;Ohnuma S;Lund PE;Pixley JN;Kimchi-Sarfaty C;Ambudkar SV;Gottesman MM
MDR 1编码的P-糖蛋白（P-gp）的药物外排功能可受到基因多态性的影响，包括MDR 1编码区的两个同义改变。在这里，我们报告的构象P-gp和它的药物外排活性可以改变同义多态性在稳定的上皮细胞单层表达P-gp。开发了几种具有相似MDR 1 DNA拷贝数的细胞系，并将其命名为LLC-MDR 1-WT（表达野生型P-gp）、LLC-MDR 1 - 3 H（表达常见单倍型P-gp）和LLC-MDR 1 - 3 HA（在3435位携带不同缬氨酸密码子的突变体）。这些细胞系表达相似水平的重组mRNA和蛋白质。在每种情况下，P-gp定位于极化细胞的顶端表面。然而，单倍型和其突变体P-GPS折叠不同于野生型，如通过UIC 2抗体移位测定和有限的蛋白水解测定所确定的。表面生物素化实验表明，非野生型P-GPS具有更长的再循环时间。药物转运试验表明，野生型和单倍型的P-gp不同的P-gp抑制剂，阻止罗丹明-123或米托蒽醌外排。此外，细胞毒性试验显示，在用P-gp抑制剂处理后，LLC-MDR 1 - 3 H细胞比LLC-MDR 1-WT细胞对米托蒽醌更具抗性。然而，多态性P-gp的表达不影响宿主细胞的形态、生长速率或单层形成。此外，ATP酶活性测定表明，无论是基础还是药物刺激的ATP酶活性的影响，在变体P-GPS。总之，我们的研究结果表明，“沉默”多态性显着改变P-gp功能，这将是预期影响个体间的药物处置和反应。
The drug efflux function of P-glycoprotein (P-gp) encoded by MDR1 can be influenced by genetic polymorphisms, including two synonymous changes in the coding region of MDR1. Here we report that the conformation of P-gp and its drug efflux activity can be altered by synonymous polymorphisms in stable epithelial monolayers expressing P-gp. Several cell lines with similar MDR1 DNA copy number were developed and termed LLC-MDR1-WT (expresses wild-type P-gp), LLC-MDR1-3H (expresses common haplotype P-gp), and LLC-MDR1-3HA (a mutant that carries a different valine codon in position 3435). These cell lines express similar levels of recombinant mRNA and protein. P-gp in each case is localized on the apical surface of polarized cells. However, the haplotype and its mutant P-gps fold differently from the wild-type, as determined by UIC2 antibody shift assays and limited proteolysis assays. Surface biotinylation experiments suggest that the non-wild-type P-gps have longer recycling times. Drug transport assays show that wild-type and haplotype P-gp respond differently to P-gp inhibitors that block efflux of rhodamine-123 or mitoxantrone. In addition, cytotoxicity assays show that the LLC-MDR1-3H cells are more resistant to mitoxantrone than the LLC-MDR1-WT cells after being treated with a P-gp inhibitor. Expression of polymorphic P-gp, however, does not affect the host cell’s morphology, growth rate, or monolayer formation. Also, ATPase activity assays indicate that neither basal nor drug-stimulated ATPase activities are affected in the variant P-gps. Taken together, our findings indicate that “silent” polymorphisms significantly change P-gp function, which would be expected to affect interindividual drug disposition and response.