Single Quantum Dot Tracking Unravels Agonist Effects on the Dopamine Receptor Dynamics,Biochemistry

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Single Quantum Dot Tracking Unravels Agonist Effects on the Dopamine Receptor Dynamics
Biochemistry ( IF 2.9 ) Pub Date : 2020-06-25 , DOI: 10.1021/acs.biochem.0c00360
Oleg Kovtun ₁ , Ruben Torres _{1,

2} , Riley S Ferguson ₁ , Travis Josephs ₃ , Sandra J Rosenthal _{1,

2,

4,

5,

6}

Affiliation

D2 dopamine receptors (DRD2s) belong to a family of G protein-coupled receptors that modulate synaptic dopaminergic tone via regulation of dopamine synthesis, storage, and synaptic release. DRD2s are the primary target for traditional antipsychotic medications; dysfunctional DRD2 signaling has been linked to major depressive disorder, attention-deficit hyperactivity disorder, addiction, Parkinson’s, and schizophrenia. DRD2 lateral diffusion appears to be an important post-translational regulatory mechanism; however, the dynamic response of DRD2s to ligand-induced activation is poorly understood. Dynamic imaging of the long isoform of DRD2 (D2L) fused to an N-terminal antihemagglutinin (HA) epitope and transiently expressed in HEK-293 cells was achieved through a combination of a high-affinity biotinylated anti-HA antigen-binding fragment (Fab) and streptavidin-conjugated quantum dots (QD). Significant reduction (∼40%) in the rate of lateral diffusion of QD-tagged D2L proteins was observed under agonist (quinpirole; QN)-stimulated conditions compared to basal conditions. QN-induced diffusional slowing was accompanied by an increase in frequency, lifetime, and confinement of temporary arrest of lateral diffusion (TALL), an intrinsic property of single receptor lateral motion. The role of the actin cytoskeleton in QN-induced diffusional slowing of D2L was also explored. The observed dynamic changes appear to be a sensitive indicator of the receptor activity status and might also spatially and temporally shape the receptor-mediated downstream signaling. This dynamic information could potentially be useful in informing drug discovery efforts based on single-molecule pharmacology.

中文翻译：

单量子点追踪揭示了激动剂对多巴胺受体动力学的影响

D2 多巴胺受体 (DRD2) 属于 G 蛋白偶联受体家族，通过调节多巴胺合成、储存和突触释放来调节突触多巴胺能张力。 DRD2 是传统抗精神病药物的主要靶点；功能失调的 DRD2 信号传导与重度抑郁症、注意力缺陷多动障碍、成瘾、帕金森氏症和精神分裂症有关。 DRD2 横向扩散似乎是一种重要的翻译后调节机制；然而，DRD2 对配体诱导的激活的动态反应知之甚少。通过高亲和力生物素化抗 HA 抗原结合片段 (Fab ）和链霉亲和素共轭量子点（QD）。与基础条件相比，在激动剂（喹吡罗；QN）刺激条件下观察到 QD 标记的 D2L 蛋白的横向扩散率显着降低（~40%）。 QN 诱导的扩散减慢伴随着频率、寿命和横向扩散暂时停止 (TALL) 限制的增加，TALL 是单受体横向运动的固有特性。还探讨了肌动蛋白细胞骨架在 QN 诱导的 D2L 扩散减慢中的作用。观察到的动态变化似乎是受体活性状态的敏感指标，并且还可能在空间和时间上塑造受体介导的下游信号传导。这种动态信息可能有助于为基于单分子药理学的药物发现工作提供信息。

更新日期：2020-06-25

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