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

项目摘要 A类G蛋白偶联受体（GPCR）已被多个小组报道形成受体 异聚体并作为彼此的变构调节剂。然而，他们的存在已经成为一个 极具争议的话题。尽管受体异聚体已被令人信服地证明， 异源表达研究，它们在这些系统中的过表达，缺乏来自高表达的证据， 受体异聚体的分辨率结构，缺乏探测其存在的工具，以及功能性 在体内的重要性加剧了这种怀疑。在这个建议中，我们采用了微创的 一种引入非天然氨基酸（UAA）的方法，使用琥珀密码子抑制，在战略上， 预测形成多巴胺2A（D2）和5-羟色胺2A（2A）的异聚界面的位置 受体（R）异聚体，已牵连在精神分裂症，其中抗精神病药物靶向一个 或其他受体构成了目前药物治疗的基础。我们采用一种技术， 沿着D2 R/2AR异聚体的界面连接相互作用的残基，以定位异聚体 界面并使复合物稳定在活性或非活性状态。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， 可光活化基团的照射。UAA可以用作光亲和探针。通过照射 策略性地并入UAA，所产生的单线态卡宾与UAA交联。 与相应的受体结合以形成共价键合的二聚体。我们发现当叠氮基苯丙氨酸 UAA取代TM 5中的D2 R（Y199），在UV照射后，向2AR的反式信号传导增强， 相互作用残基2AR（F244）也在TM 5中。这些结果表明，TM 5-TM 5 界面使异聚体稳定在活性状态。然后，形成的交联复合物可以 进一步研究使用蛋白质印迹，荧光共振能量转移（FRET），和总的内部 反射荧光（TIRF）显微镜以分析异聚体的缔合。质谱 (MS)将用于同位素标记探针的准确测量，以明确证明受体 在特定残基处交联并确认形成异聚体。具有不同同位素的帕尔斯 模式类似地并入受体，并且当用未标记的UAA以 1：1混合物，可以应用具有标记探针的蛋白质的质谱检测。我们 建议扩展这一整体方法，以测试是否交联TM 5-TM 6界面的两个 受体稳定了阻止反式信号传导的界面，稳定了非活性的异聚体。 状态该项目在东北大学的Diomedes Logothetis博士的实验室进行 （药物科学）和Roman Manetsch（化学）。这个跨学科的项目 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培训。

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.