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

粘附 G 蛋白偶联受体 B3 (ADGRB3) 的亚细胞定位及其与分泌的 C1Q 样配体相互作用的位置的测定

• 批准号: 10044199
• 负责人: David Christopher Martinelli
• 金额: $ 16.4万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10044199
• 关键词: Adhesions; Affinity; Agonist; Applications Grants; Area; Auditory; Auditory Perception; BAI3 gene; Binding; Biochemical; Brain; Brain Diseases; CRISPR/Cas technology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Cochlea; Data; Databases; Demyelinations; Development; Disease; Drug Targeting; Epitopes; Etiology; Family; Foundations; Funding; Future; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Gene Expression Profile; Genes; Genome; Goals; Grant; Hyperacusis; Investigation; Knock-in; Knowledge; Labyrinth; Ligands; Link; Location; Maintenance; Malignant Neoplasms; Mediating; Multiple Sclerosis; Mus; Mutation; Myelin; Neurons; Oligodendroglia; Orphan; Outer Hair Cells; Pain; Pathway interactions; Physiological; Protein Family; Proteins; Public Health; Publications; Research; Sensory; Signal Transduction; Synapses; Synaptic Cleft; Synaptic Transmission; Testing; Therapeutic; Virus; Work; cell type; design; extracellular; genetic association; innovation; member; neuropsychiatric disorder; novel; oligodendrocyte progenitor; pain perception; programs; receptor; receptor binding; response; small molecule; sound; spatiotemporal; stem cells; success; synaptogenesis; targeted treatment; therapeutic target; tool

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.

总结/摘要 ADGRB 3（又名ADGRB 3）BAI 3）是一种G蛋白偶联受体（GPCR），主要在大脑中表达， 是一个成员的一个鲜为人知的一类GPCR称为粘附GPCR。基因突变和SNP ADGRB 3与神经精神疾病和癌症有关，这表明该基因具有很高的价值。 治疗靶点然而，尚未提出关于ADGRB 3功能的机制解释 来解释这些基因关联先前的工作已经鉴定了C1 QL分泌蛋白质家族， ADGRB 3的高亲和力细胞外配体。由这种配体-GPCR结合诱导的信号是 未知长期目标是发现ADGRB 3-C1 QL相互作用在大脑中的功能，然后 将这些知识用于治疗。虽然Adgrb 3和C1 ql家族的基因表达模式 虽然已经在小鼠中描述过，但它们的蛋白质亚细胞定位几乎完全未知。为了 产生关于其功能的可检验假设，ADGRB 3-C1 QL相互作用的确切位置 需要确定。因此，本申请的目的是确定亚细胞的 ADGRB 3和相关C1 QL配体的定位。初步研究确定了两种可能的， ADGRB 3和C1 QL可能共定位的全新细胞环境，将对其进行研究 在这个提案中更详细。假设ADGRB 3将由神经元表达， 定位于突触间隙，并将结合从不同的相邻细胞表达的C1 QL 1。拟议 研究是创新的，因为它将1）使用尖端的CRISPR/Cas9技术， 免疫定位和2）是第一个彻底表征这些蛋白质的亚细胞位置。 所研究的每个细胞环境将以病毒介导的CRISPR组分的引入为特征， 敲入表位标签，精确追踪ADGRB 3及其C1 QL 1的亚细胞定位 配体，这将揭示关于ADGRB 3的关键时空细节，并建立工具和途径， 进一步的功能研究。这一研究意义重大，因为它将形成病原学和生物化学 为后续研究ADGRB 3信号转导障碍奠定了基础，并证明了 背景/疾病可能受益于靶向ADGRB 3-C1 QL 1结合的小分子药物 接口.它还将为今后的赠款申请提供必要的数据。未来的研究将集中于 证明了这些受体-配体相互作用在它们各自的细胞中的功能后果， 背景，并充分阐明ADGRB 3信号传导机制（预期是可推广的）。的 长期目标是设计激动剂或拮抗剂，靶向ADGRB 3-C1 QL 1结合界面， 与至少一种存在ADGRB 3的细胞环境相关的潜在治疗。