Abstract
Protein function is mainly modulated by dynamic reversible or irreversible post-translational modifications. Among them, the identification of protein phosphorylation sites and changes in phosphorylation levels in vivo are of considerable interest for a better understanding of the protein function. Thus, effective strategies for the quantitative determination of phosphorylation degrees for low abundant proteins, for which antibodies are not available, are required in order to evaluate the functional regulation of proteins attributed to phosphorylation. In this study, we used the heart β1-adrenergic receptor (Adrb1) as a model protein and developed FLAG-Adrb1 knock-in mice, in which the FLAG tag was inserted at the N-terminus of Adrb1. The phosphorylation sites and levels of Adrb1 in the heart were elucidated by immuno-affinity purification followed by quantitative mass spectrometry analysis using ion intensity ratio of the phosphorylated peptide versus corresponding unphosphorylated peptide. The phosphorylation levels at Ser274 and Ser462 of Adrb1 were approximately 0.25 and 0.0023. This effective strategy should be useful for not only analyzing site-specific phosphorylation levels of target proteins, but also quantifying the expression levels of proteins of interest when appropriate antibodies are not available.
Highlights
Phosphorylation is the most extensively studied post-translational modification (PTM) regulating protein activity and plays a pivotal role in cellular signaling events and metabolic processes
We demonstrated the effectiveness of KI mice for determining protein phosphorylation levels at specific sites for a low abundant protein of interest
The process of activating G-protein coupled receptors (GPCRs) by agonist stimulation, followed by receptor internalization and trafficking events, is well documented; little of this process has been examined in vivo
Summary
Phosphorylation is the most extensively studied post-translational modification (PTM) regulating protein activity and plays a pivotal role in cellular signaling events and metabolic processes. G-protein coupled receptors (GPCRs) are phosphorylated at multiple sites, acting as a barcode for regulating signaling in a tissue-specific manner through a combination of phosphorylation sites [1,2]. As for the study of GPCRs, protein antibodies are powerful tools for understanding GPCRs by allowing expression levels and phosphorylation states to be evaluated. Commercially available antibodies do not frequently work due to the low-specificity of antibodies and the low expression level of GPCRs in vivo. To overcome these difficulties, we have established a strategy to analyze protein phosphorylation levels at specific sites in vivo using the GPCR β1-adrenergic receptor (Adrb1) as a model protein for which all commercially available antibodies do not work
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call