title: Investigating the phosphorylation of free fatty acid receptor 4 and free fatty acid receptor 2
Creators: name: Dong, Zhaoyang
Publisher: name: University of Glasgow
Year: 2024

description: G protein-coupled receptors (GPCRs), as a large receptor family, are involved in many physiological and pathological processes. Almost all GPCRs are regulated by phosphorylation, which is a complex process and a key event in determining the downstream signal transduction. However, it is difficult to detect the phosphorylation status of the receptors in living individuals.

Free fatty acids are considered not only as dietary nutrients but also as signalling molecules because of their ability to activate the family of G protein-coupled free fatty acid receptors. Among the GPCRs for free fatty acids, free fatty acid receptor 4 (FFA4, also known as GPR120) is known to respond to long-chain fatty acids such as docosahexaenoic acid (DHA) and eicosapntemacnioc acid (EPA). FFA4 was found to regulate gut incretin glucagon-like peptide-1 (GLP-1) secretion in enteroendocrine L cells as well as insulin-sensitizing and antidiabetic effects of omega-3 polyunsaturated fatty acids. The therapeutic potential of FFA4 agonists is drawing great attention in the treatment of diabetes. Recent studies have also shown the anti-inflammatory effects of FFA4 in lung resident macrophages, as well as the mediation of airway smooth muscle relaxation. Therefore, it is vital to understand details of the identification and regulation of phosphorylation in FFA4 response to ligands. This thesis aimed to integrate phosphorylation sites of FFA4 by using novel phospho-specific antibodies, and applying the antibodies to probe phosphorylation in vivo. Additionally, this thesis also investigated the GPCR kinase (GRK) isoforms involved in the regulation of the phosphorylation of FFA4.

To verify phosphorylation sites of FFA4, we first characterised the phospho-site specific antibodies, anti-pThr347 and anti-pThr349/Ser350 . These antibodies were derived from phosphorylated peptide containing phosphorylation phosphate on residue Thr347 (GAILTDTSVK) and Thr349/Ser350 (ILTDTSVKRND). The antibodies identified the phosphorylated FFA4 in Flp-In TERx 293 cells transformed with human FFA4 (hFFA4), and this was stop by Lambda Protein Phosohatase. The immunoblots showed that the phosphorylation of FFA4 at Thr347 and Thr349/Ser350 was in an agonist-dependent way. We further treated the cells with agonists and antagonists and lysis the cells at different time point. The cell lysates were immunoprecipitated and analysed by western blot. The results demonstrated that phosphorylation of FFA4 was regulated by agonists and antagonists in timedependent patterns. The images from immunocytochemistry demonstrate the distribution of phosphorylated FFA4 in hFFA4 cells. These images also display FFA4 internalization with long-time exposure to agonist. Previous studies have reported that phosphorylation was regulated by GRKs and β-arrestins. Thus, hFFA4 cells were treated with GRK inhibitors to verify which GRKs are involved in mediating the phosphorylation of FFA4. Our results revealed that FFA4 phosphorylation is inhibited by GRK6 inhibitor compound 19. However, the βarrestin 2 recruitment signal was highly inhibited by a combination of compound 19 and compound 101 (GRK 2/3 inhibitor) suggesting that phosphorylation of FFA4 may be regulated by multiple GRKs. To investigate the phosphorylation profile of FFA4 in real tissues, I collected lung tissues from FFA4 wild type mice and FFA4 knock out mice, treated them with FFA4 agonist and analysed with western blot. However, translating these findings to endogenously expressed FFA4 in lung tissue was challenging and will require further studies to optimize conditions. As our lab also has the phosphor-site specific antibodies of FFA2, and FFA2 shows effects on metabolism and immune process, I then turned to study FFA2.

FFA2 is able to activated by short chain fatty acids, and widely express in cells and tissues, such as immune cells, adipose, and colon. FFA2 is related to metabolism and immune process, which makes it a potential target to obesity, type 2 diabetes and anti-inflammatory. To investigate whether phospho-site specific antibodies were able to identify the phosphorylated form of FFA2 in vivo, it is confirmed that the agonist-mediated phosphorylation of hFFA2- DREADD was identified by anti-pSer296/Ser297 and anti-Thr306/Thr310 FFA2 in cells stably expressing hFFA2-DREADD (hFFA2-DREADD means designer receptor exclusively activated by designer drug variant of huma FFA2). Attempts were made to translate the in vitro findings described above to hFFA2-DREADD expressed in mouse tissues. Our results illustrate that hFFA2-DREADD was phosphorylated at Ser296/Ser297 in white adipose tissue but not in Peyer’s patches. In contrast, hFFA2-DREADD was phosphorylated at Thr306/Thr310 in Peyer’s patches, but not in phosphorylated white adipose tissue. Meanwhile the resides were all phosphorylated in colonic epithelium. These findings provide evidence to GPCR phosphorylation bar-code theory.

In summary, this thesis showed the phosphorylation of FFA4 sites Thr347 and Thr349/Ser350 were agonist induced and play a time-dependent manner. The regulation of phosphorylation of FFA4 was mainly responsible by GRK6. As for FFA2, I showed that anti-pSer296/Ser297 and anti-Thr306/Thr310 identified the agonist-induced phosphorylation on specific sites in human FFA2-DREADD cells. moreover, FFA2 showed distinct phosphorylation profile in different tissues from hFFA2-DREADD mouse. These findings contribute to understanding the phosphorylation behaviours of FFA4 and FFA2, and confirm phospho-sites specific antibodies as probes to detect phosphorylation of the receptor. Conclusions drawn from these studies may help advance future efforts to validate the therapeutic potential of targeting FFA4 and FFA2.