Department of Microbiology and Immunology, Drexel University College of Medicine, PA 19129, United States.
Department of Psychiatry, Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, NY 10016, United States.
Pharmacol Res. 2019 May;143:48-57. doi: 10.1016/j.phrs.2019.03.003. Epub 2019 Mar 4.
Agonist-induced internalization of G protein-coupled receptors (GPCRs) is a significant step in receptor kinetics and is known to be involved in receptor down-regulation. However, the dopamine D3 receptor (D3R) has been an exception wherein agonist induces D3Rs to undergo desensitization followed by pharmacological sequestration - which is defined as the sequestration of cell surface receptors into a more hydrophobic fraction within the plasma membrane without undergoing the process of receptor internalization. Pharmacological sequestration renders the receptor in an inactive state on the membrane. In our previous study we demonstrated that a novel class of D3R agonists exemplified by SK608 have biased signaling properties via the G-protein dependent pathway and do not induce D3R desensitization. In this study, using radioligand binding assay, immunoblot or immunocytochemistry methods, we observed that SK608 induced internalization of human D3R stably expressed in CHO, HEK and SH-SY5Y cells which are derived from neuroblastoma cells, suggesting that it is not a cell-type specific event. Further, we have evaluated the potential mechanism of D3R internalization induced by these biased signaling agonists. SK608-induced D3R internalization was time- and concentration-dependent. In comparison, dopamine induced D3R upregulation and pharmacological sequestration in the same assays. GRK2 and clathrin/dynamin I/II are the key molecular players in the SK608-induced D3R internalization process, while β-arrestin 1/2 and GRK-interacting protein 1(GIT1) are not involved. These results suggest that SK608-promoted D3R internalization is similar to the type II internalization observed among peptide binding GPCRs.
激动剂诱导 G 蛋白偶联受体（GPCR）内化是受体动力学中的一个重要步骤，已知其参与受体下调。然而，多巴胺 D3 受体（D3R）是一个例外，其中激动剂诱导 D3R 脱敏，然后进行药理学隔离——这被定义为将细胞表面受体隔离到质膜内更疏水的部分，而不经历受体内化过程。药理学隔离使受体在膜上处于非活性状态。在我们之前的研究中，我们证明了一类新型的 D3R 激动剂，以 SK608 为例，通过 G 蛋白依赖途径具有偏向信号特性，并且不会诱导 D3R 脱敏。在这项研究中，我们使用放射性配体结合测定、免疫印迹或免疫细胞化学方法，观察到 SK608 诱导在 CHO、HEK 和 SH-SY5Y 细胞中稳定表达的人 D3R 内化，这些细胞源自神经母细胞瘤细胞，这表明这不是一种细胞类型特异性事件。此外，我们评估了这些偏向信号激动剂诱导 D3R 内化的潜在机制。SK608 诱导的 D3R 内化是时间和浓度依赖性的。相比之下，多巴胺在相同的测定中诱导 D3R 上调和药理学隔离。GRK2 和网格蛋白/动力蛋白 I/II 是 SK608 诱导的 D3R 内化过程中的关键分子参与者，而β-arrestin 1/2 和 GRK 相互作用蛋白 1（GIT1）不参与。这些结果表明，SK608 促进的 D3R 内化类似于肽结合 GPCR 中观察到的 II 型内化。
