Project description:Free Fatty Acid receptor 2 (FFA2) is activated by short-chain fatty acids and expressed widely, including in white adipocytes and various immune and enteroendocrine cells. Using a Designer Receptor Exclusively Activated by Designer Drugs (DREADD) variant of human FFA2 we explored the activation and phosphorylation profile of the receptor, both in a heterologous cell line and in tissues from a transgenic knock-in mouse line expressing this DREADD. FFA2 phospho-site specific antisera targeting either pSer296/pSer297 or pThr306/pThr310 provided sensitive biomarkers of both constitutive and agonist-mediated phosphorylation as well as an effective means to visualise agonist-activated receptors in situ. In white adipose tissue phosphorylation of residues Ser296/Ser297 was enhanced upon agonist activation whilst Thr306/Thr310 did not become phosphorylated. By contrast, in immune cells from Peyer’s patches Thr306/Thr310 become phosphorylated in a strictly agonist-dependent fashion. This was also true in enteroendocrine cells of the colon. The concept of phosphorylation bar-coding has centred to date on the potential for different agonists to promote distinct receptor phosphorylation patterns. Here we demonstrate that this occurs for the same agonist-receptor pairing in different patho-physiologically relevant target tissues. This may underpin why a single G protein-coupled receptor can generate different functional outcomes in a tissue-specific manner.
