Mechanisms of activation, signaling and trafficking of adhesion GPCRs GPR64 and GPR56

粘附 GPCR GPR64 和 GPR56 的激活、信号传导和运输机制

• 批准号: 9760422
• 负责人: Nariman Balenga
• 金额: $ 32.26万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9760422
• 关键词: ADGR1 gene; Address; Adenylate Cyclase; Adhesions; Amino Acids; Anabolism; Animal Model; Area; Arrestins; Asthma; Binding; Biochemical; Biological; Biophysics; Bone Development; Cell Surface Receptors; Cell physiology; Cells; Cellular Assay; Characteristics; Clathrin; Cleaved cell; Comparative Study; Cyclic AMP; Cyclic AMP Response Element; Data; Development; Disease; Down-Regulation; Elements; Endocrine; Endocrine system; Family; Family Study; Foundations; Functional disorder; Future; G protein coupled receptor kinase; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genetic; Goals; Human; Hyperparathyroidism; Hypertension; Immune system; In Vitro; Infertility; Insulin Resistance; Knowledge; Laboratories; Lead; Ligands; Light; Malignant Neoplasms; Mediating; Metabolic Diseases; Molecular; Molecular Conformation; Musculoskeletal; Musculoskeletal System; Mutation; N-terminal; Neurologic; Orphan; PTH gene; Parathyroid Adenoma; Parathyroid gland; Pathologic; Pathway interactions; Patients; Peptides; Pharmacology; Phenotype; Physiological; Physiological Processes; Physiology; Play; Process; Proteolysis; Publishing; Receptor Activation; Research; Rest; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Site; Specificity; Stimulus; Structure; Surface; Testing; Therapeutic; Therapeutic Intervention; Zebrafish; bone; bone metabolism; coated pit; comparative; design; extracellular; in vivo; inhibitor/antagonist; interest; member; mutant; novel; overexpression; peptide P; receptor; receptor internalization; recruit; small molecule; spatiotemporal; trafficking; translational study

Project Summary G protein-coupled receptors (GPCRs) transmit the extracellular stimuli into intracellular signals, by which they can orchestrate a myriad of cellular and physiological processes. GPCRs form the largest superfamily of surface receptors and their aberrant function causes diseases such as cancer, asthma, hypertension, endocrine and musculoskeletal dysfunction. Adhesion GPCRs (aGPCRs) are the second largest but the least studied family of GPCRs and have recently gained much interest due to their direct or indirect involvement in various diseases. In order to target aGPCRs therapeutically, we need to understand the mechanisms by which they are activated and the intracellular signaling cascades that they initiate. aGPCRs have an unusually long N-terminal fragment (NTF) that is cleaved during their biosynthesis but stays bound to the rest of the receptor non-covalently. The processes taking place after binding of extracellular ligands to this NTF can vary for different aGPCRs. While such interactions can stabilize a certain conformation and trigger or inhibit signaling in some aGPCRs, it can also dissociate the NTF and unmask a small tethered peptide (stalk) on the very N-terminus of the remaining receptor. We showed that the NTF of ADGRG2/GPR64, an orphan aGPCR, functions as an inhibitor and its deletion results in a receptor that is constitutively activated by its stalk. We provided compelling evidence that GPR64 regulates secretion of parathyroid hormone, a master regulator of bone metabolism by human-derived parathyroid adenoma cells. Via molecular and cellular assays, we have demonstrated that NTF-deficient GPR64 elevates cAMP levels by activating adenylyl cyclase, interacts with -arrestins, becomes ubiquitinated and internalizes via unknown mechanisms. A mutant that lacks this tethered peptide and NTF (stalk-less) is devoid of constitutive activity but responds to the exogenously added synthetic tethered peptide and interacts with - arrestins. The underlying mechanism for recruitment of -arrestins by stalk-less GPR64 in the absence of G- protein signaling is not clear. These published and preliminary data combined with our current knowledge of another closely related aGPCR, ADGRG1/GPR56 lead us to hypothesize that specific structural elements control signaling, endocytic pathways and physiological functions of these aGPCRs. To shed light on the pharmacological and physiological characteristics of these receptors, we will: (1) Determine the impact of NTF and its cleavage on the binding of tethered peptides and trafficking of GPR64 and GPR56; (2) Identify the structural motifs and intracellular regulators that control G-protein and -arrestin signaling downstream of GPR64 and GPR56 and (3) Determine the effects of GPR64 signaling and its NTF and stalk on bone development in Zebrafish, a low vertebrate model organism. These novel translational studies will greatly bolster our understanding of aGPCR pharmacology and lay the foundation for rational design of therapeutics for diseases caused by aGPCRs.

项目摘要 G蛋白偶联受体（GPCRs）将细胞外刺激转化为细胞内信号， 可以协调无数的细胞和生理过程。GPCR形成了最大的表面超家族 受体及其异常功能导致疾病，如癌症、哮喘、高血压、内分泌和 肌肉骨骼功能障碍粘附GPCR（aGPCR）是第二大但研究最少的家族， GPCR由于直接或间接参与多种疾病的发生，近年来引起了人们的广泛关注。 为了治疗aGPCR，我们需要了解它们被激活的机制 以及它们引发的细胞内信号级联。aGPCR具有异常长的N末端片段 (NTF)其在生物合成过程中被切割，但保持与受体的其余部分非共价结合。的 细胞外配体与该NTF结合后发生的过程对于不同的aGPCR可以不同。而 这种相互作用可以稳定某些构象并触发或抑制某些aGPCR中的信号传导， 也解离NTF，并在剩余的NTF的N-末端暴露一个小的拴系肽（茎）。 受体的我们发现ADGRG 2/GPR 64的NTF是一种孤儿aGPCR，其功能是抑制剂， 缺失导致受体被其茎部组成型激活。我们提供了有力的证据 GPR 64调节甲状旁腺激素的分泌，甲状旁腺激素是人源性骨代谢的主要调节因子。 甲状旁腺腺瘤细胞。通过分子和细胞分析，我们已经证明了NTF缺陷型GPR 64 通过激活腺苷酸环化酶提高cAMP水平，与β-抑制蛋白相互作用，变得泛素化， 通过未知的机制内化。缺乏这种栓系肽和NTF（无茎）的突变体是缺乏的。 的组成型活性，但响应于外源添加的合成拴系肽，并与β- 抑制素在缺乏G-抑制蛋白的情况下，无茎GPR 64募集β-抑制蛋白的潜在机制是： 蛋白质信号传导尚不清楚。这些公布的和初步的数据结合我们目前的知识， 另一个密切相关的aGPCR，ADGRG 1/GPR 56，使我们假设特定的结构元件控制， 这些aGPCR的信号传导、内吞途径和生理功能。办法揭露 这些受体的药理和生理特性，我们将：（1）确定NTF的影响 以及它对栓系肽的结合和GPR 64和GPR 56的运输的切割;（2）鉴定 控制GPR 64下游G蛋白和β-抑制蛋白信号传导的结构基序和细胞内调节因子 （3）测定GPR 64信号传导及其NTF和柄对骨发育的影响， 斑马鱼，一种低等脊椎动物模式生物。这些新的翻译研究将大大加强我们的 了解aGPCR药理学，为疾病治疗的合理设计奠定基础 由aGPCR引起。