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

项目摘要 粘附性GPCRs(AGPCRs)是GPCR超家族中第二大类，也是最神秘的一类。 最近的遗传学研究表明，编码黏附G蛋白的基因的多态性与 受体(AGPCR3)与注意力缺陷风险的增加密切相关 多动症(ADHD)。值得注意的是，在果蝇、斑马鱼和小鼠中，ADGRL3的表达受到干扰 增强了活动能力。在adgr13基因缺失的小鼠中，纹状体多巴胺水平显示增加， 提示这种跨物种的过度活跃表型可能是通过增强的多巴胺来调节的。 发信号。因此，ADGRL3代表了ADHD药物治疗和治疗的新靶点 其他涉及多巴胺失调的神经精神障碍，如精神分裂症和成瘾。 然而，就其激动剂的特性和信号特性而言，ADGRL3仍然是孤立的，其 人们对多巴胺神经传递的作用知之甚少。与其他aGPCR一样，ADGRL3包含7 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 适用于与蛋白质配体的粘附性相互作用。在当前的提案中，我们的目标是描述基本的 ADGRL3的信号特性以及它们如何被粘连蛋白配体调节。为此目的， 我们将使用一套全面的GPCR信号分析，并结合创新的磁力 模拟跨突触配体结合中潜在施加的机械力的方法 维持和调节突触的形态和组织。同时，我们将使用基因的组合 表征ADGRL3在多巴胺中作用的策略以及成像和行为方法 体内的神经传递。这一多学科方法也将成为今后研究的平台，目的是 在测试疾病突变对ADGRL3功能的影响和筛选ADGRL3调节因子时 发信号。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 ADGRL3控制的通路并确定N末端在信号和蛋白中的作用 利用(A)体外信号分析和策略ADGRL3调节多巴胺神经传递 突变，(B)一种创新的磁镊子分析，以评估机械力对 ADGRL3的活性和(C)一个果蝇行为模型来表征ADGRL3的定位和功能 ADGRL3在体内的多巴胺能神经元中的表达。2)确定跨突触配体的作用机制 通过测定Teneurin-1和Teneurin-1对ADGRL3信号和多巴胺神经传递的影响 使用上述体外和体内方法研究ADGRL3信号转导的其他配体。

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.