Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
Drug Metab Dispos. 2011 Nov;39(11):2148-54. doi: 10.1124/dmd.111.040295. Epub 2011 Aug 4.
Transporter adaptor protein PDZK1 regulates several influx transporters for xenobiotics and nutrients in small intestine, and their expression on the apical membrane is diminished in pdzk1 gene knockout [pdzk1(-/-)] mice. In the present study, we initially attempted to use pdzk1(-/-) mice to functionally identify influx transporters responsible for intestinal absorption of cimetidine. Contrary to our expectation, the plasma concentration of cimetidine after oral administration to pdzk1(-/-) mice was higher than that in wild-type mice, and the double peaks of plasma concentration found in wild-type mice were not observed in pdzk1(-/-) mice. Western blot analysis of intestinal brush-border membranes revealed that expression of breast cancer resistance protein (BCRP) but not of P-glycoprotein is reduced in pdzk1(-/-) mice. This result was compatible with the reduction of apical localization of BCRP in pdzk1(-/-) mice assessed by immunohistochemical analysis. Transcellular transport of cimetidine in the basal-to-apical direction in Madin-Darby canine kidney II (MDCKII) cells stably expressing both BCRP and PDZK1 (MDCKII/BCRP/PDZK1) was higher than that in MDCKII cells stably expressing BCRP (MDCKII/BCRP) cells. Moreover, MDCKII/BCRP/PDZK1 cells are more resistant than MDCKII/BCRP cells to the cytotoxicity of the anticancer agent 7-ethyl-10-hydroxycamptothecin (SN-38), which is a substrate of BCRP. These results were consistent with the higher expression of BCRP on apical membranes in MDCKII/BCRP/PDZK1 cells. Pull-down and immunoprecipitation studies revealed a physical interaction between BCRP and PDZK1. Taken together, these findings demonstrate that PDZK1 plays a pivotal role in the apical localization of BCRP. This is the first identification of a regulatory protein that physically interacts with and regulates BCRP in small intestine in vivo.
PDZK1 转运蛋白衔接蛋白调节小肠内几种外来物和营养物的摄取转运体，pdzk1 基因敲除 [pdzk1(-/-)] 小鼠的这些转运体在顶膜上的表达减少。在本研究中，我们最初试图使用 pdzk1(-/-) 小鼠来对负责西咪替丁肠道吸收的摄取转运体进行功能鉴定。与我们的预期相反，pdzk1(-/-) 小鼠口服西咪替丁后的血浆浓度高于野生型小鼠，而在野生型小鼠中观察到的血浆浓度双峰在 pdzk1(-/-) 小鼠中未观察到。肠刷状缘膜的 Western blot 分析显示，pdzk1(-/-) 小鼠中乳腺癌耐药蛋白 (BCRP) 的表达而非 P-糖蛋白的表达减少。这一结果与免疫组织化学分析显示的 pdzk1(-/-) 小鼠中 BCRP 的顶膜定位减少相符。在稳定表达 BCRP 和 PDZK1 的 Madin-Darby 犬肾 II (MDCKII) 细胞 (MDCKII/BCRP/PDZK1) 中，西咪替丁从基底到顶膜的跨细胞转运高于稳定表达 BCRP 的 MDCKII 细胞 (MDCKII/BCRP) 细胞。此外，与 MDCKII/BCRP 细胞相比，MDCKII/BCRP/PDZK1 细胞对抗癌剂 7-乙基-10-羟基喜树碱 (SN-38) 的细胞毒性更具抗性，SN-38 是 BCRP 的底物。这些结果与 MDCKII/BCRP/PDZK1 细胞中 BCRP 在顶膜上的更高表达一致。拉下和免疫沉淀研究表明 BCRP 与 PDZK1 之间存在物理相互作用。综上所述，这些发现表明 PDZK1 在 BCRP 的顶膜定位中起关键作用。这是首次在体内鉴定与 BCRP 相互作用并调节其在小肠中的表达的调节蛋白。
