Department of Pharmaceutics, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, New Jersey 08854, USA.
Pharm Res. 2010 Apr;27(4):589-96. doi: 10.1007/s11095-009-9983-2. Epub 2010 Feb 6.
Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs. We have previously shown that the activity of hOAT4 was down-regulated by activation of PKC and up-regulated by PDZ protein NHERF-1. Here, we investigated the mechanisms underlying such regulations.
COS-7 cells expressing hOAT4 were treated with PKC activator phorbol 12-myristate 13-acetate (PMA) or transfected with dominant negative mutants of dynamin-2 or Eps15 or transfected with NHERF-1. The internalization and the function of hOAT4 were then determined.
We showed that hOAT4 constitutively internalized from and recycled back to plasma membrane. Transfection of dominant negative mutants of dynamin-2 or Eps15 into the cells, all of which block clathrin-dependent endocytotic pathway, significantly blocked hOAT4 internalization. Treatment of cells with PMA accelerated hOAT4 internalization, whereas transfection of cells with NHERF-1 attenuated hOAT4 internalization.
Our studies demonstrated that i) hOAT4 undergoes constitutive trafficking between cell surface and intracellular compartments, ii) hOAT4 internalization partly occurs through clathrin-dependent pathway, iii) the down-regulation of hOAT4 activity by activation of PKC and the up-regulation of hOAT4 activity by NHERF-1 are mediated through alteration of hOAT4 internalization.
人有机阴离子转运蛋白 4（hOAT4）属于有机阴离子转运蛋白家族，在体内对具有重要临床意义的药物的分布起着关键作用。我们之前已经表明，PKC 的激活会下调 hOAT4 的活性，而 PDZ 蛋白 NHERF-1 则会上调 hOAT4 的活性。在此，我们研究了这些调节的机制。
用 PKC 激活剂佛波醇 12-肉豆蔻酸 13-醋酸酯（PMA）处理表达 hOAT4 的 COS-7 细胞，或用 dynamin-2 或 Eps15 的显性失活突变体转染，或用 NHERF-1 转染。然后测定 hOAT4 的内化和功能。
我们表明 hOAT4 从质膜内化并循环回质膜。将 dynamin-2 或 Eps15 的显性失活突变体转染到细胞中，所有这些突变体都阻断了网格蛋白依赖的内吞途径，显著阻止了 hOAT4 的内化。用 PMA 处理细胞加速了 hOAT4 的内化，而用 NHERF-1 转染细胞则减弱了 hOAT4 的内化。
我们的研究表明：i）hOAT4 在细胞表面和细胞内隔室之间进行组成型转运，ii）hOAT4 的内化部分通过网格蛋白依赖途径发生，iii）PKC 的激活下调 hOAT4 活性，NHERF-1 上调 hOAT4 活性是通过改变 hOAT4 的内化来介导的。
