Figure 5. Hypoxia induces a phosphorylation barcode in the absence of agonist binding.

Human embryonic kidney cells (HEK293) overexpressing β₂-AR were exposed to the β-agonist isoproterenol or 5-hour hypoxia. β₂-ARs were enriched by alprenolol for quantitative mass spectrometry analysis of phosphorylated peptides. 21% and 2% O₂ (n = 3); β-agonist (n = 2). (A) Spectrum for the pS246-containing peptide. The mass difference between the y₇ and y₈ ions is consistent with phosphorylation at S246. (C) Spectrum for the pS261- and pS262-containing peptide. The masses of the y₅, y₆, and y₇ ions are consistent with phosphorylation at S261 and S262. (E) Spectrum for the pS355- and pS356-containing peptide. The masses of the y₁₆, y₁₇, and y₁₈ ions are consistent with phosphorylation at S355 and S356. (B, D, and F) Dot plots showing abundance of each phosphorylated peptide at 21% or 2% oxygen or with β-agonist. (G and H) Expression of phosphorylated β₂-AR at S355/S356 and total β₂-AR in HUVECs with 21% or 2% oxygen (vehicle or GRK inhibitor at 125 μM). Replicate samples run on parallel gels are presented (n = 2). (I) Hypoxia-specific β-AR phosphorylation barcode with increased (pink) and decreased (blue) phosphorylation at unique sites.