Department of Pharmaceutics, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, New Jersey, 08854, USA.
AAPS J. 2019 Feb 13;21(2):30. doi: 10.1208/s12248-019-0303-4.
Organic anion transporter 3 (OAT3) plays a vital role in removing a broad variety of anionic drugs from kidney, thus avoiding their possible toxicity in the body. We earlier established that activation of protein kinase C (PKC) enhances OAT3 ubiquitination, which promotes OAT3 internalization from the cell plasma membrane to intracellular endosomes and consequent degradation. As a result, OAT3 expression and transport activity are reduced. In the current study, we discovered that protein kinase A (PKA) had an opposite effect to PKC on the regulation of OAT3. We showed that activation of PKA by Bt2-cAMP stimulated OAT3 transport activity, which was largely caused by an enhanced plasma membrane expression of the transporter, kinetically reflected as an augmented maximal transport velocity V without notable alteration in substrate-binding affinity K. Additionally, we showed that PKA activation accelerated the rate of OAT3 recycling from intracellular compartments to the plasma membrane and decelerated the rate of OAT3 degradation. We further showed that OAT3 is subjected to post-translational modification by SUMO-2 and SUMO-3 not by SUMO-1. PKA activation enhanced OAT3 SUMOylation, which was accompanied by a reduced OAT3 ubiquitination. Finally, insulin-like growth factor 1 significantly stimulated OAT3 transport activity and SUMOylation through PKA signaling pathway. In conclusion, this is the first demonstration that PKA stimulated OAT3 expression and transport activity by altering the trafficking kinetics of OAT3 possibly through the crosstalk between SUMOylation and ubiquitination. Our studies are consistent with a remote sensing and signaling model for transporters (Wu et al. in Mol Pharmacol. 79(5):795-805, 2011).
有机阴离子转运蛋白 3（OAT3）在从肾脏中去除多种阴离子药物方面发挥着重要作用，从而避免了它们在体内可能产生的毒性。我们之前的研究表明，蛋白激酶 C（PKC）的激活增强了 OAT3 的泛素化，这促进了 OAT3 从细胞质膜内化到细胞内的内体，并导致随后的降解。结果，OAT3 的表达和转运活性降低。在本研究中，我们发现蛋白激酶 A（PKA）对 OAT3 的调节作用与 PKC 相反。我们表明，Bt2-cAMP 激活 PKA 刺激 OAT3 转运活性，这主要是由于转运体在质膜上的表达增加，动力学上表现为最大转运速度 V 的增加，而对底物结合亲和力 K 没有明显改变。此外，我们表明 PKA 激活加速了 OAT3 从细胞内隔室到质膜的再循环速度，并减缓了 OAT3 的降解速度。我们进一步表明，OAT3 受到 SUMO-2 和 SUMO-3 的翻译后修饰，而不是 SUMO-1。PKA 激活增强了 OAT3 的 SUMO 化，伴随着 OAT3 泛素化的减少。最后，胰岛素样生长因子 1 通过 PKA 信号通路显著刺激 OAT3 的转运活性和 SUMO 化。总之，这是首次证明 PKA 通过改变 OAT3 的转运动力学来刺激 OAT3 的表达和转运活性，这可能是通过 SUMO 化和泛素化之间的串扰实现的。我们的研究与 Wu 等人提出的转运体远程传感和信号模型一致（Mol Pharmacol. 79(5):795-805, 2011）。
