Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, Ontario, Canada.
PLoS One. 2013 Aug 6;8(8):e70736. doi: 10.1371/journal.pone.0070736. Print 2013.
The activity of G protein-coupled receptors (GPCRs) is intricately regulated by a range of intracellular proteins, including G protein-coupled kinases (GRKs) and arrestins. Understanding the effects of ligands on these signaling pathways could provide insights into disease pathophysiologies and treatment. The dopamine D2 receptor is a GPCR strongly implicated in the pathophysiology of a range of neurological and neuropsychiatric disorders, particularly schizophrenia. Previous studies from our lab have shown the preclinical efficacy of a novel allosteric drug, 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA), in attenuating schizophrenia-like behavioural abnormalities in rodent models of the disease. As an allosteric modulator, PAOPA binds to a site on the D2 receptor, which is distinct from the endogenous ligand-binding site, in order to modulate the binding of the D2 receptor ligand, dopamine. The exact signaling pathways affected by this allosteric modulator are currently unknown. The objectives of this study were to decipher the in vivo effects, in rats, of chronic PAOPA administration on D2 receptor regulatory and downstream molecules, including GRK2, arrestin-3 and extracellular receptor kinase (ERK) 1/2. Additionally, an in vitro cellular model was also used to study PAOPA's effects on D2 receptor internalization. Results from western immunoblots showed that chronic PAOPA treatment increased the striatal expression of GRK2 by 41%, arrestin-3 by 34%, phospho-ERK1 by 51% and phospho-ERK2 by 36%. Results also showed that the addition of PAOPA to agonist treatment in cells increased D2 receptor internalization by 33%. This study provides the foundational evidence of putative signaling pathways, and changes in receptor localization, affected by treatment with PAOPA. It improves our understanding on the diverse mechanisms of action of allosteric modulators, while advancing PAOPA's development into a novel drug for the improved treatment of schizophrenia.
G 蛋白偶联受体 (GPCRs) 的活性受到一系列细胞内蛋白的复杂调节，包括 G 蛋白偶联激酶 (GRKs) 和抑制蛋白。了解配体对这些信号通路的影响可以深入了解疾病的病理生理学和治疗方法。多巴胺 D2 受体是一种 GPCR，强烈参与多种神经和神经精神疾病的病理生理学，特别是精神分裂症。我们实验室的先前研究表明，新型变构药物 3(R)-[(2(S)-吡咯烷羰基)氨基]-2-氧代-1-吡咯烷乙酰胺 (PAOPA) 在减弱疾病啮齿动物模型中的精神分裂样行为异常方面具有临床前疗效。作为一种变构调节剂，PAOPA 与 D2 受体上的一个结合位点结合，该结合位点与内源性配体结合位点不同，从而调节 D2 受体配体多巴胺的结合。这种变构调节剂影响的确切信号通路目前尚不清楚。本研究的目的是破译慢性 PAOPA 给药对大鼠体内 D2 受体调节和下游分子（包括 GRK2、抑制蛋白-3 和细胞外受体激酶 (ERK)1/2）的体内影响。此外，还使用体外细胞模型研究了 PAOPA 对 D2 受体内化的影响。Western 免疫印迹结果显示，慢性 PAOPA 处理使纹状体中的 GRK2 表达增加了 41%，抑制蛋白-3 增加了 34%，磷酸化 ERK1 增加了 51%，磷酸化 ERK2 增加了 36%。结果还表明，在细胞中加入 PAOPA 可使激动剂处理后的 D2 受体内化增加 33%。这项研究提供了潜在信号通路和受体定位变化的基础证据，这些变化受 PAOPA 治疗的影响。它增进了我们对变构调节剂的不同作用机制的理解，同时推进了 PAOPA 的开发，将其作为一种新型药物用于改善精神分裂症的治疗。
