title: Concomitant action of structural elements and receptor phosphorylation determines arrestin-3 interaction with the free fatty acid receptor FFA4.
abstract: In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implicated in the regulation of metabolic and inflammatory responses. Here we show, using mass spectrometry, mutagenesis, and phosphospecific antibodies, that agonist-regulated phosphorylation of the human FFA4 receptor occurred primarily at five residues (Thr(347), Thr(349), Ser(350), Ser(357), and Ser(360)) in the C-terminal tail. Mutation of these residues reduced both the efficacy and potency of ligand-mediated arrestin-3 recruitment as well as affecting recruitment kinetics. Combined mutagenesis of all five of these residues was insufficient to fully abrogate interaction with arrestin-3, but further mutagenesis of negatively charged residues revealed additional structural components for the interaction with arrestin-3 within the C-terminal tail of the receptor. These elements consist of the acidic residues Glu(341), Asp(348), and Asp(355) located close to the phosphorylation sites. Receptor phosphorylation thus operates in concert with structural elements within the C-terminal tail of FFA4 to allow for the recruitment of arrestin-3. Importantly, these mechanisms of arrestin-3 recruitment operate independently from Gq/11 coupling, thereby offering the possibility that ligands showing stimulus bias could be developed that exploit these differential coupling mechanisms. Furthermore, this provides a strategy for the design of biased receptors to probe physiologically relevant signaling.
authors.affiliation: From the Medical Research Council Toxicology Unit, University of Leicester, Hodgkin building, Lancaster Road, Leicester LE1 9HN, United Kingdom.
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Butcher
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom, and.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Hudson
authors.affiliation: Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Bharat
authors.initials: B
authors.lastname: Shimpukade
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom, and.
authors.forename: Elisa
authors.initials: E
authors.lastname: Alvarez-Curto
authors.affiliation: From the Medical Research Council Toxicology Unit, University of Leicester, Hodgkin building, Lancaster Road, Leicester LE1 9HN, United Kingdom.
authors.forename: Rudi
authors.initials: R
authors.lastname: Prihandoko
authors.affiliation: Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Trond
authors.initials: T
authors.lastname: Ulven
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom, and Graeme.Milligan@glasgow.ac.uk.
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
authors.affiliation: From the Medical Research Council Toxicology Unit, University of Leicester, Hodgkin building, Lancaster Road, Leicester LE1 9HN, United Kingdom, tba@le.ac.uk.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Tobin
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019390:Arrestins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D019390:Arrestins; C519853:FFAR4 protein, human; D043562:Receptors, G-Protein-Coupled; C082333:arrestin3
keywords: Arrestin; Diabetes; Fatty Acid; G Protein; G Protein-coupled Receptor (GPCR); Phosphorylation