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    • To validate the identified phosphorylation sites in the

    2023-10-12

    To validate the identified phosphorylation sites in the mouse heart, we analyzed HEK 293T T7 High Yield Cy5 RNA transfected with Adrb1 based on the hypothesis that protein residues phosphorylated both in vivo and in vitro are more likely to be physiologically relevant. All of the phosphorylation sites identified in the mouse heart were also identified in human cells overexpressing Adrb1, with the exception of Ser280, as shown in the sequence alignment in Fig. 3. These conserved phosphorylation sites might provide important insights into Adrb1 signaling-mediated mechanisms and drug discovery. However, we also identified phosphorylated Adrb1 residues in Adrb1-overexpressing cells that were not identified in mouse hearts (Table 1). For example, the peptide corresponding to positions 407–431 in Adrb1 (AGPPPSPGAPSDDDDDDAGTTPPAR) was triply phosphorylated at Ser412, Ser417, and Thr426/Thr427 (the exact position could not be identified) or dually phosphorylated at a combination of two of these residues. These findings might be an artifact of excessive ARDB1 levels. Alternatively, these sites might have been phosphorylated at levels too low to detect in the mouse heart. In either case, phosphorylation sites identified in vivo are considered to be more reliable. We also examined Adrb1 phosphorylation in Adrb1-overexpressing cells using SILAC labeled-peptides as an internal standard. In consequence, we observed a 2-fold increase in phosphorylation levels at Ser401 and Ser462 in ISO-stimulated cells compared with control cells. These were agonist-dependent phosphorylation sites. Although phosphorylation levels at Ser274 and Ser280 strongly increased in ISO-stimulated mouse hearts, those levels were similar in Adrb1-overexpressing cells compared with control cells (Table 1). We assume that the cell culture medium was supplemented with fetal bovine serum (FBS), and the constituent in FBS might have induced continuous Adrb1 activation. The agonist-induced phosphorylation sites identified both in vivo and in vitro, Ser462, might have essential biological functions, and their participation in signal transduction pathways merits further investigation. To investigate the kinases that mediate Adrb1 phosphorylation, we used the kinase–substrate interaction prediction algorithm NetworKIN, which can identify putative kinases based on consensus sequence motifs and physical protein-protein interactions [18]. As predicted by the NetworKIN search, the agonist-dependent phosphorylation sites in the C-terminus of Adrb1 (Ser417, Ser450, Ser451, and Ser462) are primarily mediated by GPCR kinases (GRKs). GRKs are acidotropic kinases with an affinity to acidic amino acids in the proximity of phosphorylatable residues [19]. In this respect, it is reasonable that acidic residues are enriched in the C-terminus of Adrb1. There are two types of β-ARs: Adrb1 and Adrb2. Adrb1 predominantly localizes in the heart and plays a pivotal role in the regulation of heart function, e. g. rate, force, and speed of contraction of the heart [20]. In fact, only one phosphorylated peptide fragment corresponding to positions 458–466 of Adrb2, FHAQNLpSQVEQDGR, was observed in the course of exploration of Adrb1 phosphorylation. These findings might be reasonable when considering the mRNA level of β-ARs.
    Funding
    Acknowledgements We would like to thank Dr. Okubo (Takeda Pharmaceutical Company Limited) for providing cDNA encoding for mouse Adrb1. We also thank Drs. Andou and Kuboi (Takeda Pharmaceutical Company Limited) for their encouragement and helpful discussion.
    Introduction The ventral tegmental area (VTA) is a centrolateral part of the A10, the largest group comprising dopaminergic (DA) neurons in mammalian brain. The role of the VTA neuronal activity has been extensively studied in recent decades, particularly focusing on the activity of DAergic neurons, which led to the formation of the reward prediction error (RPE) model of reinforcement learning (Schultz, 1998, Schultz, 2016). However, the VTA neuronal population is heterogeneous as it consists of DAergic (∼67%), GABAergic (∼30%) and glutamatergic (∼2–3%) neurons (Nair-Roberts et al., 2008, Ungless and Grace, 2012). Thus, the VTA non-DAergic neurons are predominantly GABAergic, as opposed to the medial parts of A10, which, apart from DAergic neurons, contain predominantly glutamatergic cells (Yamaguchi et al., 2015).