Barros Scott A, Srimaroeng Chutima, Perry Jennifer L, Walden Ramsey, Dembla-Rajpal Neetu, Sweet Douglas H, Pritchard John B
Laboratory of Pharmacology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425.
J Biol Chem. 2009 Jan 30;284(5):2672-2679. doi: 10.1074/jbc.M808078200. Epub 2008 Nov 21.
Organic anion transporters (OATs) play a pivotal role in the clearance of small organic anions by the kidney, yet little is known about how their activity is regulated. A yeast two-hybrid assay was used to identify putative OAT3-associated proteins in the kidney. Atypical protein kinase Czeta (PKCzeta) was shown to bind to OAT3. Binding was confirmed in immunoprecipitation assays. The OAT3/PKCzeta interaction was investigated in rodent renal cortical slices from fasted animals. Insulin, an upstream activator of PKCzeta, increased both OAT3-mediated uptake of estrone sulfate (ES) and PKCzeta activity. Both effects were abolished by a PKCzeta-specific pseudosubstrate inhibitor. Increased ES transport was not observed in renal slices from OAT3-null mice. Transport of the shared OAT1/OAT3 substrate, rho-aminohippurate, behaved similarly, except that stimulation was reduced, not abolished, in the OAT3-null mice. This suggested that OAT1 activity was also modified by PKCzeta, subsequently confirmed using an OAT1-specific substrate, adefovir. Inhibition of PKCzeta also blocked the increase in ES uptake seen in response to epidermal growth factor and to activation of protein kinase A. Thus, PKCzeta acted downstream of the epidermal growth factor to protein kinase A signaling pathway. Activation of transport was accompanied by an increase in V(max) and was blocked by microtubule disruption, indicating that activation may result from trafficking of OAT3 into the plasma membrane. These data demonstrate that PKCzeta activation up-regulates OAT1 and OAT3 function, and that protein-protein interactions play a central role controlling these two important renal drug transporters.
有机阴离子转运体（OATs）在肾脏清除小有机阴离子的过程中起关键作用，但关于其活性如何调节却知之甚少。采用酵母双杂交试验来鉴定肾脏中假定的与OAT3相关的蛋白。结果显示非典型蛋白激酶Czeta（PKCzeta）可与OAT3结合。免疫沉淀试验证实了这种结合。在禁食动物的啮齿动物肾皮质切片中研究了OAT3/PKCzeta的相互作用。胰岛素作为PKCzeta的上游激活剂，可增加OAT3介导的硫酸雌酮（ES）摄取以及PKCzeta活性。这两种效应均被PKCzeta特异性假底物抑制剂消除。在OAT3基因敲除小鼠的肾切片中未观察到ES转运增加。OAT1/OAT3的共同底物对氨基马尿酸的转运表现类似，只是在OAT3基因敲除小鼠中刺激作用减弱但未消除。这表明OAT1的活性也受到PKCzeta的调节，随后使用OAT1特异性底物阿德福韦得以证实。抑制PKCzeta也可阻断因表皮生长因子和蛋白激酶A激活而导致的ES摄取增加。因此，PKCzeta在表皮生长因子至蛋白激酶A信号通路的下游起作用。转运激活伴随着V(max)增加，并被微管破坏所阻断，表明激活可能是由于OAT3转运至质膜所致。这些数据表明PKCzeta激活可上调OAT1和OAT3的功能，并且蛋白质-蛋白质相互作用在控制这两种重要的肾脏药物转运体中起核心作用。
