title: Probing the stoichiometry of β2-adrenergic receptor phosphorylation by targeted mass spectrometry.
abstract: BACKGROUND
  Protein phosphorylation of G-protein-coupled receptors (GPCR) is central to the myriad of functions that these ubiquitous receptors perform in biology. Although readily addressable with the use of phospho-specific antibodies, analysis phosphorylation at the level of stoichiometry requires receptor isolation and advanced proteomics. We chose two key sites of potential phosphorylation of human beta2-adrenergic receptor (β2AR residues S355 and S356) to ascertain the feasibility of applying targeted mass spectrometry to establishing the stoichiometry of the phosphorylation.
  
  
  METHODS
  We stimulated HEK293 cells stably expressing Flag-tagged β2AR-eGFP with 10 μM beta-adrenergic agonist (isoproterenol) and made use of proteomics and targeted mass spectrometry (MS) to quantify the molar ration of phosphorylation on S355 and S356 versus non-phosphorylated receptor in agonist-treated cells.
  
  
  RESULTS
  Phosphorylation of either S355 or S356 residue occurred only for agonist-occupied β2AR. The results demonstrated that pS356 is the dominant site of protein phosphorylation. The abundance of the p356 was 8.6-fold more than that of pS355. Calculation of the molar ratio of phosphorylated (pS355 plus pS356) versus non-phosphorylated receptor reveals that at high occupancy of the receptor only 12.4% of the β2AR is phosphorylated at these sites.
  
  
  CONCLUSIONS
  Application of advanced proteomics and use of the most sensitive targeted MS strategy makes possible the detection and quantification of phosphorylation of very low abundance peptide digests of β2AR. Establishing the stoichiometry of two key sites of agonist-stimulated phosphorylation with β2AR is an essential first-step to global analysis of the stoichiometry of GPCR phosphorylation.
authors.affiliation: Department of Pharmacology, Health Sciences Center, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA. shujuan.gao@stonybrook.edu.
authors.forename: Shujuan
authors.initials: S
authors.lastname: Gao
authors.forename: Craig
authors.initials: C
authors.lastname: Malbon
authors.forename: Hsien-Yu
authors.initials: HY
authors.lastname: Wang