Determination of the subcellular localization of adhesion G protein-coupled receptor B3 (ADGRB3) and its locations of interaction with secreted C1Q-like ligands

摘要/摘要 ADGRB3(又名BAI3)是一种G蛋白偶联受体(GPCR)，主要在大脑和 是被称为粘附性GPCRs的一类鲜为人知的GPCRs的成员。基因突变和单核苷酸多态 ADGRB3与神经精神疾病和癌症都有关联，这表明该基因具有很高的价值 治疗靶点。然而，还没有提出关于ADGRB3功能的机械解释 来解释这些基因关联。以前的工作已经确定C1QL分泌的蛋白质家族是 ADGRB3的高亲和力胞外配体。这种配体-GPCR结合所产生的信号是 未知。长期目标是发现ADGRB3-C1QL相互作用在大脑中的功能，然后 利用这些知识获得治疗收益。虽然Adgrb3和C1ql家族的基因表达谱 已经在小鼠身上描述过，但它们的蛋白质亚细胞定位几乎完全未知。为了 生成关于其功能、ADGRB3-C1QL相互作用的确切位置的可测试假设 需要确定。因此，这一应用的目标是确定亚细胞 ADGRB3及其相关的C1QL配体的定位。初步研究确定了两种可能的和 ADGRB3和C1QL可能共定位的全新细胞环境，这将被研究 在这项提案中有更详细的说明。假设ADGRB3将由神经元和 定位于突触裂隙，并将与不同相邻细胞表达的C1QL1结合。建议数 研究具有创新性，因为它将1)使用尖端的CRISPR/Cas9技术来促进 免疫定位和2)第一个彻底确定这些蛋白质的亚细胞位置。 每个被研究的细胞环境都将以病毒介导的CRISPR组件的引入为特色 在表位标签敲击中，允许精确追踪ADGRB3及其C1QL1的亚细胞定位 Ligand，它将揭示关于ADGRB3的关键时空细节，并建立工具和途径 进一步的功能研究。这项研究具有重要的意义，因为它将形成病原学和生化 为后续研究ADGRB3信号的紊乱奠定了基础，并证明了哪些细胞 环境/疾病可能受益于靶向ADGRB3-C1QL1结合的小分子药物 界面。它还将为未来的赠款申请提供必要的数据。未来的研究将集中在 展示了这些受体-配体相互作用在各自细胞中的功能后果 上下文，并充分阐明ADGRB3信号机制(预计可推广)。这个 长期目标是设计激动剂或拮抗剂，将ADGRB3-C1QL1结合界面作为靶点 潜在的治疗与ADGRB3存在的至少一个细胞环境有关。

SUMMARY/ABSTRACT ADGRB3 (a.k.a. BAI3) is a G protein-coupled receptor (GPCR) that is predominantly expressed in brain and is a member of a poorly understood class of GPCRs known as adhesion GPCRs. Mutations and SNPs in ADGRB3 are linked to both neuropsychiatric diseases and cancers, suggesting this gene is a high value therapeutic target. However, no mechanistic explanations regarding ADGRB3 function have been proposed to explain these genetic associations. Previous work has identified the C1QL secreted family of proteins as the high-affinity extracellular ligands for ADGRB3. The signals induced by this ligand-GPCR binding are unknown. The long-term goal is to discover the functions of ADGRB3-C1QL interactions in the brain, then to use this knowledge for therapeutic gain. Although gene expression patterns of Adgrb3 and the C1ql family have been described in mice, their protein subcellular localizations are almost entirely unknown. In order to generate testable hypotheses regarding their function, the exact locations of ADGRB3-C1QL interaction need to be determined. Therefore, the objective of this application is to determine the subcellular localization of ADGRB3 and an associated C1QL ligand. Preliminary studies identify two possible and completely novel cellular contexts where ADGRB3 and a C1QL may co-localize, which will be investigated in greater detail in this proposal. The hypothesis is that ADGRB3 will be expressed by neurons and localized to the synaptic cleft, and will bind to C1QL1 expressed from distinct adjacent cells. The proposed research is innovative because it will 1) use cutting edge CRISPR/Cas9 technology to facilitate immunolocalization and 2) be the first to thoroughly characterize the subcellular locations of these proteins. Each cellular context investigated will feature virus-mediated introduction of CRISPR components resulting in the knockin of epitope tags allowing precise tracking of subcellular localization of ADGRB3 and its C1QL1 ligand, which will reveal critical spatiotemporal details about ADGRB3 and establish tools and a pathway for further functional study. This research is significant because it will form the etiological and biochemical foundation for subsequent research on disorders of ADGRB3 signaling, and demonstrate which cellular contexts/diseases might benefit from a small molecule drug targeting the ADGRB3-C1QL1 binding interface. It will also provide prerequisite data for future grant applications. Future research will focus on demonstrating the functional consequences of these receptor-ligand interactions in their respective cellular contexts, and fully elucidating ADGRB3 signaling mechanisms (which is expected to be generalizable). The longer-term goal is to design agonists or antagonists to target the ADGRB3-C1QL1 binding interface as a potential therapy related to at least one of the cellular contexts where ADGRB3 is present.