Hafkemeyer P, Dey S, Ambudkar S V, Hrycyna C A, Pastan I, Gottesman M M
Laboratory of Cell Biology, Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892, USA.
Biochemistry. 1998 Nov 17;37(46):16400-9. doi: 10.1021/bi980871+.
P-glycoprotein (Pgp), the product of the MDR1 gene, confers multidrug resistance on cancer cells by ATP-dependent extrusion of anticancer drugs. Biochemical and genetic studies with Pgp have identified the putative transmembrane (TM) region 12 (residues 974-994) as a major region involved in drug interactions with amino acid residues conserved among Pgp family members shown to be essential for transport. To determine whether nonconserved residues might be involved in substrate specificity, seven amino acid residues were identified within TM 12 that were not strictly conserved among the MDR1 and MDR2 family of proteins from different mammalian species. We replaced all seven of these amino acid residues with alanine, one at a time and in combinations, and used a vaccinia virus based transient expression system to analyze function. None of the single replacements caused any alteration in transport function. However, when residues L975, V981, and F983 were replaced collectively, drug transport, drug-stimulated ATP hydrolysis, and photoaffinity labeling with the drug analogue, [125I]iodoarylazidoprazosin (IAAP), were abrogated, with little effect on [alpha-32P]-8-azido-ATP labeling and basal ATPase activity. Pairwise alanine substitutuions showed variable effects on function. Substitutions including L975A in combination with any one of the other two replacements had the least effect on Pgp function. The V981A and F983A double mutant showed the most effect on transport of fluorescent substrates. In contrast, alanine substitutions of all four nonconserved residues M986, V988, Q990, and V991 at the putative carboxy-terminal half of TM 12 showed no effect on drug transport except for a partial reduction in bodipy-verapamil extrusion. These results suggest that nonconserved residues in the putative amino-proximal half of TM 12 of Pgp play a more direct role in determining specificity of drug transport function than those in the putative carboxy-terminal half of TM 12.
多药耐药基因1（MDR1）的产物P-糖蛋白（Pgp）通过依赖ATP的抗癌药物外排赋予癌细胞多药耐药性。对Pgp进行的生化和遗传学研究已确定推定的跨膜（TM）区域12（第974 - 994位氨基酸残基）是与药物相互作用的主要区域，该区域中Pgp家族成员间保守的氨基酸残基对转运至关重要。为了确定非保守残基是否可能参与底物特异性，在TM 12内鉴定出七个氨基酸残基，它们在来自不同哺乳动物物种的MDR1和MDR2蛋白家族中并非严格保守。我们将这七个氨基酸残基逐个及组合地替换为丙氨酸，并使用基于痘苗病毒的瞬时表达系统分析其功能。单个替换均未引起转运功能的任何改变。然而，当第975位亮氨酸（L975）、第981位缬氨酸（V981）和第983位苯丙氨酸（F983）残基被一起替换时，药物转运、药物刺激的ATP水解以及用药物类似物[125I]碘芳叠氮基哌唑嗪（IAAP）进行的光亲和标记均被消除，而对[α-32P]-8-叠氮基-ATP标记和基础ATP酶活性影响很小。成对的丙氨酸替换对功能显示出不同的影响。包括L975A与其他两个替换之一组合的替换对Pgp功能影响最小。V981A和F983A双突变体对荧光底物的转运影响最大。相比之下，在TM 12推定的羧基末端一半中所有四个非保守残基第986位甲硫氨酸（M986）、第988位缬氨酸（V988）、第990位谷氨酰胺（Q990）和第991位缬氨酸（V991）的丙氨酸替换，除了硼替韦拉帕米外排略有减少外，对药物转运没有影响。这些结果表明，Pgp的TM 12推定的氨基近端一半中的非保守残基在决定药物转运功能的特异性方面比TM 12推定的羧基末端一半中的非保守残基发挥更直接的作用。
