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Strategy for Determination of β 1-Adrenergic Receptor Phosphorylation State In Vivo

Kozo Hayashi and Hiroyuki Kobayashi

β1-adrenergic receptor (Adrb1), a member of the G-protein coupled receptor (GPCR) superfamily, is a critical regulator of heart function. All GPCRs are phosphorylated at multiple sites and the specific pattern of phosphorylation acts as a “barcode” to regulate receptor function and downstream physiological processes in a tissue-specific manner. However, little is known about the location and function of Adrb1 phosphorylation sites in vivo due to the lack of specific antibodies. As a first step to identify the phosphorylation states of Adrb1 and associated functions in the in vivo mouse heart, we developed the following experimental strategy: 1) identification of agonist-dependent Adrb1 phosphorylation sites in isolated perfused mouse heart using advanced phosphoproteomics techniques; 2) definitive assignment of these phosphorylation sites by high-quality mass spectrometry (MS) data obtained from Adrb1- overexpressing HEK 293T cells; 3) generation of knock-in (KI) Mice expressing Adrb1 fused with FLAG-tag at the N-terminus for immunoaffinity purification to reveal phosphorylation status within the living organism; 4) elucidation of phosphorylation levels at specific sites of Adrb1 in KI mouse heart by MS measures of phosphorylated peptide to corresponding unphosphorylated peptide ion intensity ratios. Using this strategy, we identified Ser462 at the C-terminus of Adrb1 as an agonist-dependent phosphorylation site in the perfused mouse heart. We also revealed the basal phosphorylation ratios at Ser274 (0.25), Ser417 (0.55) and Ser462 (0.0023) in KI Mice. These findings provide novel insights into the regulatory mechanisms of Adrb1 function mediated by site-specific phosphorylation.