Mann Anika, Keen Alastair C, Mark Hanka, Dasgupta Pooja, Javitch Jonathan A, Canals Meritxell, Schulz Stefan, Robert Lane J
Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany.
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
Sci Rep. 2021 Apr 15;11(1):8288. doi: 10.1038/s41598-021-87417-2.
The dopamine D receptor (DR) is the target of drugs used to treat the symptoms of Parkinson's disease and schizophrenia. The DR is regulated through its interaction with and phosphorylation by G protein receptor kinases (GRKs) and interaction with arrestins. More recently, DR arrestin-mediated signaling has been shown to have distinct physiological functions to those of G protein signalling. Relatively little is known regarding the patterns of DR phosphorylation that might control these processes. We aimed to generate antibodies specific for intracellular DR phosphorylation sites to facilitate the investigation of these mechanisms. We synthesised double phosphorylated peptides corresponding to regions within intracellular loop 3 of the hDR and used them to raise phosphosite-specific antibodies to capture a broad screen of GRK-mediated phosphorylation. We identify an antibody specific to a GRK2/3 phosphorylation site in intracellular loop 3 of the DR. We compared measurements of DR phosphorylation with other measurements of DR signalling to profile selected DR agonists including previously described biased agonists. These studies demonstrate the utility of novel phosphosite-specific antibodies to investigate DR regulation and signalling.
多巴胺 D 受体（DR）是用于治疗帕金森病和精神分裂症症状的药物靶点。DR 通过与 G 蛋白受体激酶（GRK）相互作用并被其磷酸化以及与阻遏蛋白相互作用来进行调节。最近研究表明，DR 阻遏蛋白介导的信号传导具有与 G 蛋白信号传导不同的生理功能。关于可能控制这些过程的 DR 磷酸化模式，人们所知相对较少。我们旨在生成针对细胞内 DR 磷酸化位点的特异性抗体，以促进对这些机制的研究。我们合成了与 hDR 细胞内环 3 内区域相对应的双磷酸化肽，并使用它们来产生磷酸化位点特异性抗体，以广泛筛选 GRK 介导的磷酸化。我们鉴定出一种针对 DR 细胞内环 3 中 GRK2/3 磷酸化位点的特异性抗体。我们将 DR 磷酸化的测量结果与 DR 信号传导的其他测量结果进行比较，以分析选定的 DR 激动剂，包括先前描述的偏向激动剂。这些研究证明了新型磷酸化位点特异性抗体在研究 DR 调节和信号传导方面的实用性。
