Unnatural Amino Acid Receptor Incorporation as a Novel Photoaffinity Tool for GPCR Heteromer Signaling Studies

非天然氨基酸受体掺入作为 GPCR 异聚体信号研究的新型光亲和工具

基本信息

批准号：
10441230
负责人：
Brenda Teall Winn
金额：
$ 4.08万
依托单位：
NORTHEASTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10441230
关键词：
Amber Amino Acid Receptors Amino Acids Amino Acyl Transfer RNA Amino Acyl-tRNA Synthetases Antipsychotic Agents Azides Binding Biochemical Biological Assay Cells Chemistry Codon Nucleotides Complement Complex Coupled Coupling Detection Dimerization Dopamine Dopamine D2 Receptor Electrodes Environment Fingerprint Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Functional disorder G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs Heterodimerization In Vitro Isoleucine Isotope Labeling Isotopes Label Leucine Link Literature Maps Mass Spectrum Analysis Measurement Measures Methods Molecular Molecular Conformation Mutagenesis Mutate Pattern Peptides Pharmaceutical Chemistry Pharmacologic Substance Pharmacotherapy Phenylalanine Photoaffinity Labels Positioning Attribute Proteins Receptor Signaling Reporter Reporting Research Resolution Sampling Schizophrenia Science Scientist Serotonin Side Signal Transduction Site Site-Directed Mutagenesis Structure System Tail Techniques Terminator Codon Testing Training Transfer RNA Tyrosine Ultraviolet Rays Universities Validation Western Blotting biophysical chemistry biophysical techniques carbene covalent bond crosslink dimer improved in vivo irradiation minimally invasive nitrene novel overexpression photoactivation prevent protein complex protein protein interaction receptor receptor function reconstitution release of sequestered calcium ion into cytoplasm tool ultraviolet irradiation unnatural amino acids unpublished works voltage clamp

项目摘要

PROJECT SUMMARY Class A G protein-coupled receptors (GPCRs) have been reported by multiple groups to form receptor heteromers and serve as allosteric regulators of one another. Yet their mere existence has become a highly contested topic. Even though receptor heteromers have been convincingly demonstrated in heterologous expression studies, their overexpression in these systems, a lack of evidence from high resolution structures of receptor heteromers, the lack of tools to probe their existence, and functional importance in vivo have fueled this skepticism. In this proposal, we employ a minimally invasive approach of introducing unnatural amino acids (UAAs), using amber codon suppression, at strategic positions predicted to form the heteromeric interface of the dopamine 2A (D2) and serotonin 2A (2A) receptor (R) heteromer that has been implicated in schizophrenia, where antipsychotics targeting one or the other receptor form the current basis of pharmacotherapy. We employ a technique to covalently link interacting residues along the interface of the D2R/2AR heteromer in order to map the heteromer interface and stabilize the complex in the active or inactive state. Photoaffinity labeling (PAL) is a technique to investigate binding interactions by forming a covalent bond between two entities via irradiation of a photoactivatable group. UAAs can function as photoaffinity probes. By irradiation of the strategically incorporated UAA, the singlet carbene that is produced is crosslinked with the corresponding receptor to form a covalently bonded dimer. We show that when an azido-phenylalanine UAA replaces D2R(Y199) in TM5, trans-signaling to 2AR is potentiated upon UV irradiation, as does its interacting residue 2AR(F244) that is also in the TM5. These results suggest that the TM5-TM5 interface stabilizes the heteromer in the active state. The crosslinked complex that is formed can then be further studied using western blot, fluorescence resonance energy transfer (FRET), and total internal reflection fluorescence (TIRF) microscopy to analyze association of the heteromer. Mass spectrometry (MS) will be used for accurate measurement of isotopically labeled probes to definitively prove receptor crosslinking at specific residues and to confirm formation of a heteromer. PALs with a distinct isotope pattern are similarly incorporated into receptors, and when prepared with the non-labeled UAAs in a 1:1 mixture, a mass spectrometric detection of proteins with the labeled probe can be applied. We propose to expand this overall approach to test whether crosslinking the TM5-TM6 interface of the two receptors stabilizes an interface that prevents trans-signaling, stabilizing the heteromer in the inactive state. This project is conducted at Northeastern University in the labs of Drs. Diomedes Logothetis (Pharmaceutical Sciences) and Roman Manetsch (Chemistry). This interdisciplinary project of molecular biophysics and medicinal chemistry will allow for rigorous training in both environments.

项目摘要多组已经报道了A类G蛋白偶联受体（GPCR）以形成受体异域并作为彼此的变构调节器。然而，他们的存在已成为一个竞争激烈的话题。即使受体异构体在异源表达研究，它们在这些系统中的过表达，缺乏来自高的证据受体异构体的分辨率结构，缺乏探测其存在的工具以及功能体内的重要性加剧了这种怀疑。在此提案中，我们采用了最低侵入性的使用琥珀色密码子抑制引入非天然氨基酸（UAA）的方法预测形成多巴胺2A（D2）和5-羟色胺2A（2A）的异质体界面的位置与精神分裂症有关的受体（R）异构体，其中抗精神病药靶向一个或其他受体构成了药物治疗的当前基础。我们采用一种协调的技术链接沿D2R/2AR异构体界面界面的相互作用残基，以映射异符界面并稳定在活动状态或无活性状态下的复合物。光性标签（PAL）是通过在两个实体之间形成共价键来研究结合相互作用的技术光活化组的辐照。 UAAS可以用作光性探针。通过辐照策略性地纳入UAA，生产的单线碳连接与相应的受体形成共价键合二聚体。我们表明，氮杂苯丙氨酸 UAA在TM5中取代D2R（Y199），在紫外线照射下将变速箱降低到2AR，它的变速相互作用的残基2AR（F244）也位于TM5中。这些结果表明TM5-TM5 界面稳定在活性状态下的异构体。然后形成的交联成件可以使用蛋白质印迹，荧光共振能量转移（FRET）和总内部进一步研究反射荧光（TIRF）显微镜以分析异构体的关联。质谱法（MS）将用于精确测量同位素标记的探针，以确定证明受体在特定残基上进行交联，并确认形成异构体。伴侣具有独特的同位素类似地将模式纳入受体，当用未标记的UAA制备时 1：1混合物，可以应用与标记探针的蛋白质的质谱检测。我们提议扩展这种总体方法，以测试是否连接两个TM5-TM6接口受体稳定了一种防止反式信号的界面，使异构体在非活性中稳定状态。该项目是在东北大学的博士实验室进行的。 Diomedes logothetis （药科学）和罗马人（化学）。这个跨学科项目分子生物物理学和药物化学将允许在这两种环境中进行严格的训练。