Phosphorylation bar-coding of free fatty acid receptor 2 is generated in a tissue-specific manner

Free fatty acid receptor 2 (FFAR2) is activated by short-chain fatty acids and expressed widely, including in white adipocytes and various immune and enteroendocrine cells. Using both wild-type human FFAR2 and a designer receptor exclusively activated by designer drug (DREADD) variant we explored the activation and phosphorylation profile of the receptor, both in heterologous cell lines and in tissues from transgenic knock-in mouse lines expressing either human FFAR2 or the FFAR2-DREADD. FFAR2 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 whilst in enteroendocrine cells of the colon both Ser²⁹⁶/Ser²⁹⁷ and Thr³⁰⁶/Thr³¹⁰ were poorly phosphorylated. 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.