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

基本信息

批准号：
10163210
负责人：
JOHN A. OLSON
金额：
$ 31.01万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10163210
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

项目摘要

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（GPCR）将细胞外刺激传递到细胞内信号中，通过它们可以编排无数的细胞和物理过程。 GPCR构成表面最大的超家族受体及其异常功能会导致癌症，哮喘，高血压，内分泌和肌肉骨骼功能障碍。粘附GPCR（AGPCR）是第二大但最少的研究家族 GPCR并因其直接或间接参与各种疾病而引起了很多兴趣。为了热靶向AGPCR，我们需要了解激活它们的机制以及它们启动的细胞内信号传导级联。 AGPCR具有异常长的N末端片段（NTF）在其生物合成过程中切割，但无外交与其他受体结合。在细胞外配体与该NTF结合后发生的过程可能会因不同的AGPCR而异。尽管这种相互作用可以稳定某些AGPCR中的某种构象并触发或抑制信号传导，它可以还可以分离NTF，并在其余的N末端揭开小链球肽（茎）受体。我们表明，孤儿AGPCR的ADGRG2/GPR64的NTF充当抑制剂及其它的NTF 删除导致接收器始终被其茎激活。我们提供了令人信服的证据 GPR64调节甲状旁腺甲酮的分泌，甲状旁腺的骨骼代谢的主要调节剂甲状旁腺腺瘤细胞。通过分子和细胞测定，我们已经证明了NTF缺陷的GPR64 通过激活腺苷酸环化酶（与-rarstins相互作用）来提高营地水平，成为泛素化和通过未知机制进行内部化。缺少这种束缚肽和NTF（无茎）的突变体是无孔的本构活性的却反应于外源添加的合成束缚肽，并与-相互作用逮捕蛋白。在没有g-的情况下，通过无茎的GPR64募集-rarstin的基本机制蛋白质信号不清楚。这些已发布和初步数据与我们当前的知识相结合另一个密切相关的AGPCR ADGRG1/GPR56导致我们假设特定的结构元素控制这些AGPCR的信号传导，内吞通路和物理功能。阐明这些受体的药理和物理特征，我们将：（1）确定NTF的影响它的裂解对束缚的辣椒的结合以及GPR64和GPR56的贩运；（2）确定控制GPR64下游的G蛋白和-arrestin信号的结构基序和细胞内调节剂 GPR56和（3）确定GPR64信号传导及其NTF的影响以及茎对骨骼发育中的影响斑马鱼，一种低脊椎动物模型生物。这些新颖的翻译研究将极大地加强我们的了解AGPCR药理学并为疾病治疗理性设计奠定基础由AGPCR引起。