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DOI	10.1073/pnas.2024146118
	High-mass MALDI-MS unravels ligand-mediated G protein-coupling selectivity to GPCRs
	Wu N.; Olechwier A.M.; Brunner C.; Edwards P.C.; Tsai C.-J.; Tate C.G.; Schertler G.F.X.; Schneider G.; Deupi X.; Zenobi R.; Ma P.
发表日期	2021
ISSN	0027-8424
卷号	118 期号:31
英文摘要	G protein-coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, we developed a high-sensitivity, high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to interrogate the first stage of signal transduction. GPCR-G protein complex formation is detected as a proxy for the effect of ligands on GPCR conformation and on coupling selectivity. Over 70 ligand-GPCR-partner protein combinations were studied using as little as 1.25 pmol protein per sample. We determined the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [β1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). We found that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. We exemplified our quantification method using β1AR and demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. We also quantified complex formation with wild-type heterotrimeric Gαiβγ and β-arrestin-1 and showed that carvedilol induces an increase in coupling of β-arrestin-1 and Gαiβγ to β1AR. A normalization strategy allows us to quantitatively measure the binding affinities of GPCRs to partner proteins. We anticipate that this methodology will find broad use in screening and characterization of GPCR-targeting drugs. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	Coupling selectivity; G protein-coupled receptor; G proteins; MALDI mass spectrometry; Protein-protein interaction
语种	英语
scopus关键词	beta arrestin 1; guanine nucleotide binding protein; ligand; Nb80 nanobody; opiate receptor; protein binding; retina S antigen; single chain fragment variable antibody; animal; chemistry; gene expression regulation; genetics; HEK293 cell line; human; matrix-assisted laser desorption-ionization mass spectrometry; metabolism; molecular model; mouse; procedures; protein conformation; turkey (bird); Animals; Arrestin; beta-Arrestin 1; Gene Expression Regulation; GTP-Binding Proteins; HEK293 Cells; Humans; Ligands; Mice; Models, Molecular; Protein Binding; Protein Conformation; Receptors, Opioid; Single-Chain Antibodies; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Turkeys
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/251091
作者单位	Department of Chemistry and Applied Biosciences, ETH Zürich, Zürich, CH-8093, Switzerland; Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen PSI, CH-5232, Switzerland; Department of Biology, ETH Zürich, Zürich, CH-8093, Switzerland; Medical Research Council Laboratory of Molecular Biology, Cambridge, CB2 0QH, United Kingdom; Condensed Matter Theory Group, Paul Scherrer Institute, Villigen PSI, CH-5232, Switzerland
推荐引用方式 GB/T 7714	Wu N.,Olechwier A.M.,Brunner C.,et al. High-mass MALDI-MS unravels ligand-mediated G protein-coupling selectivity to GPCRs[J],2021,118(31).
APA	Wu N..,Olechwier A.M..,Brunner C..,Edwards P.C..,Tsai C.-J..,...&Ma P..(2021).High-mass MALDI-MS unravels ligand-mediated G protein-coupling selectivity to GPCRs.Proceedings of the National Academy of Sciences of the United States of America,118(31).
MLA	Wu N.,et al."High-mass MALDI-MS unravels ligand-mediated G protein-coupling selectivity to GPCRs".Proceedings of the National Academy of Sciences of the United States of America 118.31(2021).