Middle-Down Mass Spectrometry Reveals Activity-Modifying Phosphorylation Barcode in a Class C G Protein-Coupled Receptor.

Autor:	Ives AN; Department of Chemistry, Northwestern University, Evanston, Illinois 60208 United States., Dunn HA; Department of Neuroscience, The Scripps Research Institute, Jupiter, Florida 33458, United States.; Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba R3E 0T6, Canada.; Division of Neurodegenerative Disorders, St. Boniface Hospital Albrechtsen Research Centre, Winnipeg, Manitoba R2H 2A6, Canada., Afsari HS; Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States., Seckler HDS; Department of Chemistry, Northwestern University, Evanston, Illinois 60208 United States., Foroutan MJ; Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States., Chavez E; Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States., Melani RD; Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States.; National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, Illinois 60208, United States., Fellers RT; National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, Illinois 60208, United States., LeDuc RD; National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, Illinois 60208, United States., Thomas PM; Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States.; National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, Illinois 60208, United States., Martemyanov KA; Department of Neuroscience, The Scripps Research Institute, Jupiter, Florida 33458, United States., Kelleher NL; Department of Chemistry, Northwestern University, Evanston, Illinois 60208 United States.; Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States.; National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, Illinois 60208, United States., Vafabakhsh R; Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States.
Jazyk:	angličtina
Zdroj:	Journal of the American Chemical Society [J Am Chem Soc] 2022 Dec 21; Vol. 144 (50), pp. 23104-23114. Date of Electronic Publication: 2022 Dec 07.
DOI:	10.1021/jacs.2c10697
Abstrakt:	G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans. They mediate nearly all aspects of human physiology and thus are of high therapeutic interest. GPCR signaling is regulated in space and time by receptor phosphorylation. It is believed that different phosphorylation states are possible for a single receptor, and each encodes for unique signaling outcomes. Methods to determine the phosphorylation status of GPCRs are critical for understanding receptor physiology and signaling properties of GPCR ligands and therapeutics. However, common proteomic techniques have provided limited quantitative information regarding total receptor phosphorylation stoichiometry, relative abundances of isomeric modification states, and temporal dynamics of these parameters. Here, we report a novel middle-down proteomic strategy and parallel reaction monitoring (PRM) to quantify the phosphorylation states of the C-terminal tail of metabotropic glutamate receptor 2 (mGluR2). By this approach, we found that mGluR2 is subject to both basal and agonist-induced phosphorylation at up to four simultaneous sites with varying probability. Using a PRM tandem mass spectrometry methodology, we localized the positions and quantified the relative abundance of phosphorylations following treatment with an agonist. Our analysis showed that phosphorylation within specific regions of the C-terminal tail of mGluR2 is sensitive to receptor activation, and subsequent site-directed mutagenesis of these sites identified key regions which tune receptor sensitivity. This study demonstrates that middle-down purification followed by label-free quantification is a powerful, quantitative, and accessible tool for characterizing phosphorylation states of GPCRs and other challenging proteins.
Databáze:	MEDLINE
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