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reconstitution release of sequestered calcium ion into cytoplasm tool ultraviolet irradiation unnatural amino acids unpublished works voltage clamp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

项目总结