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
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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.
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.