Evaluation of signalling and phosphorylation responses of the human histamine H₄ receptor (H₄R) and the human free fatty acid receptor 4 (FFA4)

The knowledge that G protein-coupled receptors (GPCRs) are regulated by phosphorylation in a process that results in the recruitment of arrestins, leading to receptor desensitisation is now well known. The histamine H₄ receptor (H₄R) and the free fatty acid receptor 4 (FFA4) are family A GPCRs that both have the ability to become phosphorylated in their third intracellular loops and C-terminal tails by kinases found in the cytosolic milieu of the cells and tissues they are expressed in. Investigations into the phosphorylation status of the histamine H4 receptor have revealed a receptor that is highly phosphorylated even in the basal state. The endogenous, full agonist for the human H₄R, histamine, induced a robust increase in receptor phosphorylation. However, the β-arrestin-biased agonist JNJ7777120 did not. Extending this study using mass spectrometry revealed the individual sites of phosphorylation. Histamine and JNJ7777120 also caused H₄R internalisation. Our data suggests a similar level of endocytosis induced by histamine and JNJ7777120 at 5 or 30 min stimulation. Thus, we show that JNJ7777120, while previously demonstrating its differing effects on H₄R signalling, also shows differences in the phosphorylation of the H₄R when compared to histamine. Using the wild type FFA4 receptor and its phosphorylation-deficient mutants, we show the importance of phosphorylation in the recruitment of arrestin to the receptor as well as delineating G protein-dependent and independent downstream signalling pathways. Knowledge of the different signalling cascades and their mechanism of activation would be useful in the design of biased ligands for therapeutic benefits in order to develop safer and more efficacious drugs. The use of a FFA4 receptor which is phosphorylation-deficient and, therefore, couples to arrestin-3 in a reduced manner may be useful in proof-of-concept studies where the downstream signalling in a physiological setting is mediated by arrestin as opposed to G proteins. Further evidence of the importance of phosphorylation is provided by my work with the phosphomimetic FFA4 mutant receptor, which I show does not faithfully mimic phosphorylated serine and/or threonine residues resulting, in a reduced ability to couple to arrestin-3.