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

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.