Modulation of dopaminergic neurotransmission by ADGRL3, an adhesion GPCR associated with ADHD susceptibility

项目摘要 粘附GPCR（aGPCR）形成GPCR超家族的第二大但最神秘的类别。 最近的遗传学研究表明，编码粘附G蛋白偶联蛋白的基因多态性， 受体（aGPCR）亲脂蛋白3（ADGRL 3）与注意力缺陷的风险增加密切相关 多动症（ADHD）。值得注意的是，在果蝇、斑马鱼和小鼠中，ADGRL 3表达的破坏 增强的自发活动。在adgrl 3缺失小鼠中，纹状体多巴胺水平显示增加， 这表明跨物种的多动表型可能是通过增强多巴胺介导的， 信号因此，ADGRL 3代表了用于开发ADHD药物治疗的新靶点， 其他涉及多巴胺失调的神经精神疾病，如精神分裂症和成瘾。 然而，ADGRL 3在其激动剂的身份和信号传导特性方面仍然是孤儿，并且其在细胞内的表达与细胞内的表达无关。 多巴胺神经传递的作用知之甚少。与其他aGPCR一样，ADGRL 3含有7个 跨膜结构域和包含蛋白质结构域阵列的细胞外N-末端结构 适合于与蛋白质配体的粘附相互作用。在目前的提案中，我们的目标是描述基本的 ADGRL 3的信号传导特性以及它们如何被粘附蛋白配体调节。为此目的 我们将使用一套全面的GPCR信号分析与创新的磁力相结合， 用于模拟在跨突触配体结合中潜在施加的机械力的测定，所述跨突触配体的功能是 维持和调节突触形态和组织。与此同时，我们将使用基因的组合， 结合成像和行为方法来表征ADGRL 3在多巴胺 体内神经传递这一多学科方法也将作为未来研究的平台， 在测试疾病突变对ADGRL 3的功能影响和筛选ADGRL 3的调节剂方面， 信号为了实现这些目标，我们提出了以下具体目标：1）鉴定G蛋白 ADGRL 3控制的信号通路，并建立N末端在信号传导和 使用（a）体外信号传导测定和策略性ADGRL 3调节多巴胺神经传递 突变，（B）一种创新的磁镊子测定，以评估机械力对突变的影响， （c）用于表征ADGRL 3的定位和功能的果蝇行为模型， 体内多巴胺能神经元中的ADGRL 3。2)为了确定跨突触配体 通过确定teneurin-1的作用调节ADGRL 3信号传导和多巴胺神经传递， 使用上述体外和体内方法，研究其他配体对ADGRL 3信号传导的影响。

Project Summary Adhesion GPCRs (aGPCRs) form the second largest, yet most enigmatic class of the GPCR superfamily. Recent genetic findings show that polymorphisms in the gene encoding the adhesion G protein-coupled receptor (aGPCR) latrophilin 3 (ADGRL3) are strongly associated with an increased risk of attention deficit hyperactivity disorder (ADHD). Remarkably, in fruit flies, zebrafish and mice, disruption of ADGRL3 expression enhanced locomotor activity. In adgrl3 null mice, striatal dopamine levels were shown to be increased, suggesting that the cross-species hyperactive phenotype may be mediated through enhanced dopamine signaling. Thus, ADGRL3 represents a novel target for the development of drug treatments for ADHD and other neuropsychiatric disorders that involve dopamine dysregulation, such as schizophrenia and addiction. However, ADGRL3 remains orphan with respect to the identity of its agonists and signaling properties, and its role in dopamine neurotransmission is poorly understood. Like other aGPCRs, ADGRL3 contains a 7 transmembrane domain and an extracellular N-terminal architecture comprising an array of protein domains suitable for adhesive interactions with protein ligands. In the current proposal we aim to characterize the basic signaling properties of ADGRL3 and how they can be modulated by adhesive protein ligands. For this purpose we will use a comprehensive suite of GPCR signaling assays combined with an innovative magnetic force assay to mimic the mechanical force potentially exerted in binding of trans-synaptic ligands that function to maintain and modulate synapse morphology and organization. In parallel, we will use a combination of genetic strategies together with imaging and behavioral approaches to characterize the role of ADGRL3 in dopamine neurotransmission in vivo. This multidisciplinary approach will also serve as a platform for future studies aimed at testing the functional impact of disease mutations in ADGRL3 and in screening for modulators of ADGRL3 signaling. To achieve these goals we propose the following specific aims: 1) To identify the G protein pathways controlled by ADGRL3 and to establish the role of the N terminus in signaling and in regulating dopamine neurotransmission using (a) in vitro signaling assays and strategic ADGRL3 mutations, (b) an innovative magnetic tweezer assay to evaluate the influence of mechanical force on the activity of ADGRL3 and (c) a Drosophila behavioral model to characterize the localization and function of ADGRL3 in dopaminergic neurons in vivo. 2) To identify the mechanism by which trans-synaptic ligands modulate ADGRL3 signaling and dopamine neurotransmission by determining the effect of teneurin-1 and other ligands on ADGRL3 signaling using the in vitro and in vivo approaches described above.