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.

项目概要 G 蛋白偶联受体 (GPCR) 将细胞外刺激转化为细胞内信号，通过这些信号可以协调无数的细胞和生理过程，形成最大的表面超家族。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 (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而最近引起了人们的广泛兴趣。为了在治疗上靶向 aGPCR，我们需要了解它们被激活的机制它们启动的细胞内信号级联具有异常长的 N 端片段。 (NTF) 在生物合成过程中被裂解，但仍以非共价键方式与受体的其余部分结合。细胞外配体与该 NTF 结合后发生的过程可能因不同的 aGPCR 而异。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，它可以还解离 NTF 并揭开剩余部分 N 末端的一个小的束缚肽（茎）我们证明 ADGRG2/GPR64（一种孤儿 aGPCR）的 NTF 具有抑制剂及其受体的功能。缺失导致受体被其茎组成型激活，我们提供了令人信服的证据。 GPR64 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通过分子和细胞测定，我们已经证明 NTF 缺陷的 GPR64。通过激活腺苷酸环化酶提高 cAMP 水平，与 -arrestins 相互作用，变得泛素化并通过未知机制内化。缺乏这种束缚肽和 NTF（无茎）的突变体是缺乏的。的组成活性，但响应外源添加的合成束缚肽并与 - 相互作用在没有 G- 的情况下，无茎 GPR64 募集 -arrestins 的基本机制。这些已发表的初步数据与我们目前的知识相结合尚不清楚。另一个密切相关的aGPCR，ADGRG1/GPR56引导我们拦截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。为了了解这些受体的药理和生理特性，我们将：（1）确定NTF的影响及其裂解对束缚肽的结合以及 GPR64 和 GPR56 的运输的影响； (2) 鉴定控制 GPR64 下游 G 蛋白和 -arrestin 信号传导的结构基序和细胞内调节因子和 GPR56 以及 (3) 确定 GPR64 信号传导及其 NTF 和茎对骨发育的影响斑马鱼是一种低等脊椎动物模型生物，这些新颖的转化研究将极大地支持我们。了解aGPCR药理学，为合理设计疾病疗法奠定基础由 aGPCR 引起。