Development of a HiBiT Peptide-Based NanoBRET Ligand Binding Assay for Galanin Receptor 1 in Live Cells.

IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Hu Zhu, Harrison C Daly, Tae Gyun Yang, Josh Born, Gisela Andrea Camacho-Hernandez, Mingyang Yuan, Mari Inglese, Vinoth Kumar Chenniappan, Kris Zimmerman, Robin Hurst, Xin Hu, Amy Hauck Newman, Sergi Ferré, Rachel Friedman Ohana, Matthew D Hall, Samarjit Patnaik
{"title":"Development of a HiBiT Peptide-Based NanoBRET Ligand Binding Assay for Galanin Receptor 1 in Live Cells.","authors":"Hu Zhu, Harrison C Daly, Tae Gyun Yang, Josh Born, Gisela Andrea Camacho-Hernandez, Mingyang Yuan, Mari Inglese, Vinoth Kumar Chenniappan, Kris Zimmerman, Robin Hurst, Xin Hu, Amy Hauck Newman, Sergi Ferré, Rachel Friedman Ohana, Matthew D Hall, Samarjit Patnaik","doi":"10.1021/acschembio.5c00166","DOIUrl":null,"url":null,"abstract":"<p><p>Galanin is a neuroendocrine peptide that regulates a wide range of physiological functions, including feeding and energy homeostasis, mood and anxiety, and modulation of pain. The function of the galanin peptide is mediated through its three galanin receptors, namely, GALR1, GALR2, and GALR3, which belong to the G protein-coupled receptor family. To measure the interaction of ligands with galanin receptor 1 (GALR1) in living cells, we developed a novel HiBiT peptide-based NanoBRET ligand binding assay. We generated six bioluminescence resonance energy transfer (BRET) tracers composed of modified and truncated galanin peptide derivatives tagged with a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) acceptor fluorophore. The fluorophore-tagged peptide tracers were evaluated in cells expressing GALR1 tagged with HiBiT, an 11-amino acid subunit of NanoLuc, which, upon high affinity complementation with the cell-impermeable subunit LgBiT, reconstituted a functional NanoLuc luciferase. Addition of the furimazine substrate induced BRET to the BODIPY fluorophore acceptor component of the galanin-derived peptide tracers and produced a fluorescence signal output. Using this BRET assay, we characterized the binding affinity and binding kinetics of tracers with GALR1 in both equilibrium and real time. To validate our assay, we evaluated the binding affinity and function of a panel of unmodified galanin-derived peptide ligands through the competitive displacement of bound fluorescent galanin tracers. Our data showed that the binding affinity of these galanin peptide ligands correlated well with their rank order in β-arrestin recruitment and internalization functional assays. This study demonstrates that the HiBiT peptide-based NanoBRET ligand binding assay is a valuable system for studying the ligand engagement of GALR1 in living cells, offering an alternative to neuropeptide radioligand binding assays.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5000,"publicationDate":"2025-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Chemical Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1021/acschembio.5c00166","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

Galanin is a neuroendocrine peptide that regulates a wide range of physiological functions, including feeding and energy homeostasis, mood and anxiety, and modulation of pain. The function of the galanin peptide is mediated through its three galanin receptors, namely, GALR1, GALR2, and GALR3, which belong to the G protein-coupled receptor family. To measure the interaction of ligands with galanin receptor 1 (GALR1) in living cells, we developed a novel HiBiT peptide-based NanoBRET ligand binding assay. We generated six bioluminescence resonance energy transfer (BRET) tracers composed of modified and truncated galanin peptide derivatives tagged with a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) acceptor fluorophore. The fluorophore-tagged peptide tracers were evaluated in cells expressing GALR1 tagged with HiBiT, an 11-amino acid subunit of NanoLuc, which, upon high affinity complementation with the cell-impermeable subunit LgBiT, reconstituted a functional NanoLuc luciferase. Addition of the furimazine substrate induced BRET to the BODIPY fluorophore acceptor component of the galanin-derived peptide tracers and produced a fluorescence signal output. Using this BRET assay, we characterized the binding affinity and binding kinetics of tracers with GALR1 in both equilibrium and real time. To validate our assay, we evaluated the binding affinity and function of a panel of unmodified galanin-derived peptide ligands through the competitive displacement of bound fluorescent galanin tracers. Our data showed that the binding affinity of these galanin peptide ligands correlated well with their rank order in β-arrestin recruitment and internalization functional assays. This study demonstrates that the HiBiT peptide-based NanoBRET ligand binding assay is a valuable system for studying the ligand engagement of GALR1 in living cells, offering an alternative to neuropeptide radioligand binding assays.

基于HiBiT肽的活细胞甘丙氨酸受体1纳米obret配体结合试验的建立。
甘丙肽是一种调节多种生理功能的神经内分泌肽,包括摄食和能量稳态、情绪和焦虑以及疼痛调节。甘丙肽的功能是通过其三个甘丙肽受体介导的,即GALR1、GALR2和GALR3,它们属于G蛋白偶联受体家族。为了测量活细胞中配体与GALR1受体(GALR1)的相互作用,我们开发了一种基于HiBiT肽的NanoBRET配体结合实验。我们制备了六种生物发光共振能量转移(BRET)示踪剂,这些示踪剂由修饰和截断的甘丙肽衍生物组成,标记为4,4-二氟-4-硼-3a,4 -二氮-s-吲哚烯(BODIPY)受体荧光团。荧光团标记的肽示踪剂在表达带有HiBiT标记的GALR1的细胞中进行了评估,HiBiT是NanoLuc的11个氨基酸亚基,通过与细胞不渗透亚基LgBiT的高亲和力互补,重建了功能性NanoLuc荧光素酶。呋喃嘧啶底物的加入诱导BRET进入丙氨酸衍生肽示踪剂的BODIPY荧光基团受体组分,并产生荧光信号输出。利用这种BRET分析,我们在平衡和实时两种情况下表征了示踪剂与GALR1的结合亲和力和结合动力学。为了验证我们的实验,我们通过竞争置换结合的荧光甘丙肽示踪剂,评估了一组未经修饰的甘丙肽衍生肽配体的结合亲和力和功能。我们的数据显示,这些丙氨酸肽配体的结合亲和力与它们在β-阻滞蛋白募集和内化功能分析中的等级顺序密切相关。本研究表明,基于HiBiT肽的NanoBRET配体结合试验是研究活细胞中GALR1配体结合的有价值的系统,提供了神经肽放射配体结合试验的替代方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
ACS Chemical Biology
ACS Chemical Biology 生物-生化与分子生物学
CiteScore
7.50
自引率
5.00%
发文量
353
审稿时长
3.3 months
期刊介绍: ACS Chemical Biology provides an international forum for the rapid communication of research that broadly embraces the interface between chemistry and biology. The journal also serves as a forum to facilitate the communication between biologists and chemists that will translate into new research opportunities and discoveries. Results will be published in which molecular reasoning has been used to probe questions through in vitro investigations, cell biological methods, or organismic studies. We welcome mechanistic studies on proteins, nucleic acids, sugars, lipids, and nonbiological polymers. The journal serves a large scientific community, exploring cellular function from both chemical and biological perspectives. It is understood that submitted work is based upon original results and has not been published previously.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信