Stride B D, Valdimarsson G, Gerlach J H, Wilson G M, Cole S P, Deeley R G
Cancer Research Laboratories, Queen's University, Kingston, Canada.
Mol Pharmacol. 1996 Jun;49(6):962-71.
In vitro, overexpression of the human multidrug-resistance protein (MRP) causes a form of multidrug resistance similar to that conferred by P-glycoprotein, although the two proteins are only very distantly related. Studies with MRP-enriched membrane vesicles have demonstrated that the protein can bind and transport cysteinyl leukotrienes, as well as some other glutathione conjugates, with high affinity. In contrast, there is no direct evidence of the ability of MRP to bind or transport unmodified forms of the drugs to which it confers resistance. To facilitate studies of the physiological function(s) of MRP and its ability to cause multidrug resistance in vivo, we cloned and characterized the mRNA specifying its murine homolog. The murine MRP mRNA encodes a protein of 1528 amino acids that is 88% identical to human MRP. Although detectable by Northern blotting at variable levels in a wide range of tissues, in situ hybridization experiments revealed that MRP mRNA expression in some tissues is cell-type specific. High levels of the mRNA were detected in epithelia lining bronchi and bronchioles, as well as stage-specific expression in the seminiferous epithelium of the testes. Comparison of the predicted hydropathy profiles of human and murine MRP suggests a highly conserved membrane topology, the most distinctive feature of which is an extremely hydrophobic NH2-terminal region containing five or six potential transmembrane sequences. This structural feature is shared with the sulfonylurea receptor and the yeast cadmium factor 1 but is not present in members of the superfamily, such as the cystic fibrosis transmembrane conductance regulator and P-glycoproteins. Finally, we used overlapping cDNAs to construct an episomally replicating murine MRP expression vector that was stably transfected into HeLa cells. MRP-Transfected cell populations expressed markedly elevated levels of a 180-190-kDa protein that cross-reacted with a polyclonal antiserum raised against a peptide that is completely conserved in murine and human MRPs. The MRP transfectants also displayed increased resistance to vincristine (5-6-fold) and doxorubicin (< 2-fold).
在体外，人多药耐药蛋白（MRP）的过表达会导致一种类似于P-糖蛋白所赋予的多药耐药形式，尽管这两种蛋白的亲缘关系非常远。对富含MRP的膜囊泡的研究表明，该蛋白能够以高亲和力结合并转运半胱氨酰白三烯以及一些其他谷胱甘肽偶联物。相比之下，没有直接证据表明MRP具有结合或转运其赋予耐药性的未修饰形式药物的能力。为了便于研究MRP的生理功能及其在体内引起多药耐药的能力，我们克隆并鉴定了编码其小鼠同源物的mRNA。小鼠MRP mRNA编码一个由1528个氨基酸组成的蛋白质，该蛋白质与人类MRP的同源性为88%。尽管通过Northern印迹法在多种组织中可检测到不同水平的MRP mRNA，但原位杂交实验表明，某些组织中的MRP mRNA表达具有细胞类型特异性。在支气管和细支气管的上皮中检测到高水平的mRNA，以及在睾丸生精上皮中的阶段特异性表达。对人和小鼠MRP预测的亲水性图谱的比较表明，其膜拓扑结构高度保守，最显著的特征是一个极疏水的NH2末端区域，包含五或六个潜在的跨膜序列。这种结构特征与磺酰脲受体和酵母镉因子1共有，但不存在于超家族成员中，如囊性纤维化跨膜电导调节因子和P-糖蛋白。最后，我们使用重叠cDNA构建了一个可在游离型复制的小鼠MRP表达载体，并将其稳定转染到HeLa细胞中。转染了MRP的细胞群体表达了明显升高水平的180 - 190 kDa蛋白，该蛋白与针对在小鼠和人类MRP中完全保守的肽段产生的多克隆抗血清发生交叉反应。MRP转染细胞对长春新碱（5 - 6倍）和阿霉素（< 2倍）的耐药性也有所增加。
