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

• 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
• 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release of sequestered calcium ion into cytoplasm; tool; ultraviolet irradiation; unnatural amino acids; unpublished works; voltage clamp

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.

项目总结 A、G类蛋白偶联受体(GPCRs)已被多个群体报道形成受体 异构体，并作为彼此的变构调节剂。然而，他们的存在本身就变成了一种 争论不休的话题。尽管受体异构体已经在 异源表达研究，它们在这些系统中的过度表达，缺乏来自高 受体异构体的拆分结构，缺乏探测其存在的工具，以及功能 在体内的重要性加剧了这种怀疑。在这项建议中，我们采用了一种微创 在战略上引入非天然氨基酸(UAA)的方法，使用琥珀密码子抑制 预测形成多巴胺2A(D2)和5-羟色胺2A(2A)异构体界面的位置 与精神分裂症有关的受体(R)异构体，其中抗精神病药物针对一种 或者另一种受体构成了目前药物治疗的基础。我们采用了一种技术来共价 连接D2R/2AR异构体界面上的相互作用残基以映射异构体 并将复合体稳定在激活或非激活状态。光亲和标记(PAL)是一种 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 对可光活化基团的照射。尿酸可作为光亲和探针发挥作用。通过照射 战略上结合了UAA，生产的单线态卡宾与 相应的受体形成共价键合的二聚体。我们证明了当叠氮-苯丙氨酸 UAA取代了TM5中的D2R(Y199)，向2AR的反式信号在紫外线照射下被增强，其 相互作用残基2AR(F244)，也在TM5中。这些结果表明，TM5-TM5 界面使异构体稳定在活性状态。然后，形成的交联物可以 进一步利用蛋白质印迹、荧光共振能量转移(FRET)和总内 反射荧光(TIRF)显微镜分析异构体的缔合情况。质谱学 (MS)将用于准确测量同位素标记的探针，以明确证明受体 在特定残基上进行交联，并确认异构体的形成。有着不同同位素的朋友 模式类似地被结合到受体中，并且当与非标记UAA一起制备时 1：1混合物，可以用标记的探针进行蛋白质的质谱学检测。我们 建议扩展这一整体方法来测试是否将TM5-TM6接口交叉连接 受体稳定防止反式信号转导的界面，稳定处于不活跃状态的异构体 州政府。这个项目是在东北大学Diomedes Logothetis博士的实验室进行的 (药学)和罗曼·马内奇(化学)。这项跨学科的项目 分子生物物理学和药物化学将允许在这两个环境中进行严格的培训。