Identification Of Phosphorylated Residues In The Mouse FFAR4 (mGPR120) Receptor And Their Role In Beta-arrestin-2 Recruitment

E Alvarez-Curto1, R Prihandoko2, A Butcher2, B Hudson1, AB Tobin2, G Milligan1. 1University Of Glasgow, Glasgow, UK, 2University Of Leicester, Leicester, UK

The long chain free fatty acid (LCFA) receptor FFAR4 (GPR120) has the potential to regulate insulin secretion and control inflammatory processes related to metabolic syndrome (1). This 7TM receptor is widely expressed in pancreatic cells, adipose tissue and macrophages where it responds to LCFAs including α-linolenic acid and other omega-3 (n-3) polyunsaturated FAs. FFAR4 can signal in a bimodal fashion through coupling to either the heterotrimeric G-proteins, Gq/11, or through interactions with β-arrestins (2). The latter requires receptor phosphorylation and may lead to distinct signalling pathways with certain anti-inflammatory effects linked specifically to β-arrestin interaction. Conversely, stimulation of GLP-1 release has been associated with calcium release through G-protein coupling. Therefore, there is great interest in establishing the nodes and divergences between the two pathways and potentially to identify biased agonists for therapeutic use.

This study aimed to define the patterns of agonist-induced receptor phosphorylation and relate this to receptor signalling, e.g. β-arrestin recruitment and to develop pharmacological and molecular tools to better understand the biology of the mouse FFA4 (mFFA4) receptor in a physiolo-gical context.

The selective synthetic agonist TUG-891 ([(3-(4-((4-fluoro-4’-methyl-[1,1’-biphenyl]-2-yl)methoxy)phenyl)propanoic acid]) (3) promoted phosphorylation of threonines 347 and 349, and serine 350 within the carboxyl terminal tail of the receptor as identified by mass spectrometry in CHO cells transiently expressing a HA-tagged mFFA4 construct. These residues were systematically mutated to alanine to study their contribution to β-arrestin-2 recruitment and to downstream signalling pathways. Using these mutants we identified differences in efficacy (reduced by up to 70-80%) on agonist-dependent β-arrestin-2 recruitment without change in potency for the agonist (Table). The kinetics of receptor-β-arrestin-2 interaction were assessed in HEK293 cells transiently co-transfected with the mutated mFFA4 receptors linked to eYFP and a Renilla luciferase tagged β-arrestin-2 in a bioluminescence resonance energy transfer (BRET)-based assay. From these kinetic assays the half-time of recruitment for each mutant was calculated (Table).

In summary, we identified key sites of phosphorylation and residues important for agonist-dependent β-arrestin-2 recruitment. This is central to defining the contribution of modifications such as phosphorylation to the interaction interface between the receptor and β-arrestin-2 molecule and in understanding the physiological consequences of altering these sites.

References

(1) Oh et al., (2010), (2) Ragagopal et al., (2010), (3) Hudson et al., (2013)