Roles of transporter adaptors as regulatory mechanism for drug absorption and disposition

目前已鉴定出多种类型的异种转运蛋白。它们通常表现出对各种底物的多特异性识别，并介导治疗剂的膜渗透，从而在药物吸收和处置中发挥重要作用。我们最近根据体外实验数据提出，涉及异种转运蛋白的蛋白质-蛋白质相互作用可能会影响它们的功能、在质膜上的定位和表达。直接与转运蛋白相互作用的所谓接头蛋白包括PDZ(PSD95)、Dig和含有Zoo结构域的蛋白。该项目旨在阐明这种接头蛋白在体内的药代动力学作用。利用pdzk1基因敲除(pdzk1^+)小鼠，发现与PDZ接头pdzk1的相互作用对于三种溶质载体，slc15a1(寡肽转运体PEPTD，S1c22a5(肉碱/有机阳离子转运体OCTn2)和slco 1a(有机ANI…)的细胞表面定位是必不可少的更多关于多肽在小鼠小肠中的转运。电子显微镜显示PEPT1定位于pdzk1^+小鼠细胞内的囊泡结构。在pdzk1^+小鼠中，与野生小鼠相比，PEPT1底物头孢氨辛和OCTN2底物肉碱的胃肠吸收延迟。此外，pdzk1^+小鼠小肠顶膜对OATP1a底物--雌酮的摄取也减少。因此，PDZK1作为转运蛋白的调节者发挥着关键作用，从而影响这些相互作用的转运蛋白对底物的吸收。由于PDZ适配器的结构中有多个PDZ结构域，并且每个PDZ结构域都可以与转运蛋白的胞浆区域相互作用，因此可以推测转运蛋白定位于由几个转运蛋白和适配器组成的网络中。我们还发现，在小GTP结合蛋白Rab8(Rab8^+)基因敲除小鼠中，PEPT1和SLC5a1(钠/葡萄糖共转运体，SGLT1)的顶端定位几乎完全减少。胃肠对PEPT1的底物甘氨酰肌氨酸和SGLT1的底物α-甲基葡萄糖的胃肠摄取在Rab8^+中伴随减少。因此，我们的结果表明，Rab8对于两个转运蛋白的正确定位和作为这些转运蛋白底物的各种营养物质的消化是必要的。较少

Many types of xenobiotic transporters have been identified. They generally exhibit multispecific recognition of various types of substrates, and mediate membrane permeation of therapeutic agents, thereby playing important roles in drug absorption and disposition. We have recently proposed that protein-protein interactions involving the xenobiotic transporters may affect their function, localization and expression on plasma membranes based on in vitro experimental data. So-called adaptor proteins that directly interact with the transporters include PDZ (PSD95, Dig and ZOO domain-containing proteins. This project was performed with an aim to clarify pharmacokinetic roles of such adaptor proteins in vivo. Using pdzk1 gene knockout (pdzk1^+) mice, it was found that interaction with a PDZ adaptor PDZK1 is essential for the cell-surface localization of three solute carriers, Slc15a1 (oligopeptide transporter PEPTD, S1c22a5 (carnitine/organic cation transporter OCTN2) and Slco 1a (organic ani … More on transporting polypeptide, OATP1A) in mouse small intestine. Electron microscopy revealed localization of PEPT1 in intracellular vesicular structures in pdzk1^+ mice. In pdzk1^+ mice, gastrointestinal absorption of cephalexin, a substrate of PEPT1 and carnitine, a substrate of OCTN2 was delayed, compared with wild mice. In addition, uptake of estrone sulfate, a substrate of OATP1A from apical membrane of small intestine was also decreased in pdzk1^+ mice. Thus, PDZK1 plays pivotal roles as a regulator of transporters, thereby affecting the absorption of substrates of those interacting transporters. Since PDZ adaptors have multiple PDZ domains in their structure, and each PDZ domain can interact with the cytosolic region of the transporters, it can be speculated that transporters are localized within networks consisting of several transporters and adaptors. We have also found that apical localization of PEPT1 and Slc5a1 (sodium/glucose cotransporter, SGLT1) was almost completely reduced in gene knockout mice for small GTP-binding protein rab8 (rab8^+). Gastrointestinal uptake across the apical membranes of glycylsarcosine, a substrate of PEPT1 and α-methylglucose, a substrate of SGLT1 was concomitantly reduced in rab8^+. Thus, our results demonstrate that rab8 is necessary for the proper localization of the two transporters and digestion of various nutrients which are the substrate of those transporters. Less