Currier S J, Kane S E, Willingham M C, Cardarelli C O, Pastan I, Gottesman M M
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1992 Dec 15;267(35):25153-9.
The human MDR1 gene encodes the multidrug transporter (P-glycoprotein), a multidrug efflux pump. The highly homologous MDR2 gene product does not appear to be a functional multidrug pump. We have constructed a chimeric protein in which the first intracytoplasmic loop and the third and fourth transmembrane domains of the MDR1 protein were replaced by the analogous region of MDR2. Substitution of the MDR2 sequences encompassing amino acid residues 140 to 229 resulted in 17 amino acid changes, 10 in the intracytoplasmic loop (amino acids 141-188) and 7 in the transmembrane regions. This chimeric protein was expressed on the surface of NIH 3T3 cells where it bound [3H]azidopine but did not confer drug resistance. When only 4 residues, 165, 166, 168, and 169, were changed back to MDR1 amino acids, a functional drug transporter was recovered. When residues 165, 166, 168, and 169 from MDR2 were substituted into a functional MDR1 cDNA, the resulting construction was not able to confer drug resistance. These results indicate that the major functional differences between MDR1 and MDR2 in this region of P-glycoprotein reside in a small segment of the first intracytoplasmic loop. We also independently analyzed the effect of replacing Asn183 of MDR1 with Ser which occurs in MDR2. Substitution of Ser at position 183 in combination with Val at position 185 in P-glycoprotein resulted in a relative increase in resistance to actinomycin D, vinblastine, and doxorubicin in transfected NIH 3T3 cells. These results emphasize the importance of the first intracytoplasmic loop in P-glycoprotein in determining function and relative drug specificity of the transporter.
人类多药耐药基因1（MDR1）编码多药转运体（P-糖蛋白），一种多药外排泵。高度同源的多药耐药基因2（MDR2）产物似乎不是一种功能性多药泵。我们构建了一种嵌合蛋白，其中MDR1蛋白的第一个胞质内环以及第三和第四个跨膜结构域被MDR2的类似区域所取代。替换包含氨基酸残基140至229的MDR2序列导致17个氨基酸发生变化，10个在胞质内环（氨基酸141 - 188），7个在跨膜区域。这种嵌合蛋白表达于NIH 3T3细胞表面，在那里它能结合[³H]叠氮哌啶，但不赋予耐药性。当仅4个残基，即165、166、168和169变回MDR1氨基酸时，恢复了功能性药物转运体。当将MDR2的残基165、166、168和169替换到功能性MDR1 cDNA中时，所得构建体无法赋予耐药性。这些结果表明，P-糖蛋白这一区域中MDR1和MDR2之间的主要功能差异存在于第一个胞质内环的一小段中。我们还独立分析了将MDR1的天冬酰胺183替换为MDR2中出现的丝氨酸的影响。在P-糖蛋白中，将183位的丝氨酸与185位的缬氨酸组合替换，导致转染的NIH 3T3细胞对放线菌素D、长春碱和阿霉素的耐药性相对增加。这些结果强调了P-糖蛋白中第一个胞质内环在决定转运体功能和相对药物特异性方面的重要性。
