title: Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein-Coupled Receptor 120.
abstract: It is established that long-chain free fatty acids includingω-3 fatty acids mediate an array of biologic responses through members of the free fatty acid (FFA) receptor family, which includes FFA4. However, the signaling mechanisms and modes of regulation of this receptor class remain unclear. Here, we employed mass spectrometry to determine that phosphorylation of mouse (m)FFAR4 occurs at five serine and threonine residues clustered in two separable regions of the C-terminal tail, designated cluster 1 (Thr(347), Thr(349), and Ser(350)) and cluster 2 (Ser(357)and Ser(361)). Mutation of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment of arrestin 3, receptor internalization, and activation of Akt were regulated by mFFA4 phosphorylation. The analysis of mFFA4 phosphorylation-dependent signaling was extended further by selective mutations of the phosphoacceptor sites. Mutations within cluster 2 did not affect agonist activation of Akt but instead significantly compromised receptor internalization and arrestin 3 recruitment. Distinctly, mutation of the phosphoacceptor sites within cluster 1 had no effect on receptor internalization and had a less extensive effect on arrestin 3 recruitment but significantly uncoupled the receptor from Akt activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode) at the C terminus of the receptor.
authors.affiliation: Medical Research Council Toxicology Unit, University of Leicester, Leicester, United Kingdom (R.P., A.J.B., A.B.T.); Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology (E.A.-C., B.D.H., G.M.), and Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary, and Life Sciences (A.M.M.), University of Glasgow, Glasgow, United Kingdom; and Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Odense, Denmark (T.U.).
authors.forename: Rudi
authors.initials: R
authors.lastname: Prihandoko
authors.affiliation: Medical Research Council Toxicology Unit, University of Leicester, Leicester, United Kingdom (R.P., A.J.B., A.B.T.); Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology (E.A.-C., B.D.H., G.M.), and Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary, and Life Sciences (A.M.M.), University of Glasgow, Glasgow, United Kingdom; and Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Odense, Denmark (T.U.).
authors.forename: Elisa
authors.initials: E
authors.lastname: Alvarez-Curto
authors.affiliation: Medical Research Council Toxicology Unit, University of Leicester, Leicester, United Kingdom (R.P., A.J.B., A.B.T.); Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology (E.A.-C., B.D.H., G.M.), and Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary, and Life Sciences (A.M.M.), University of Glasgow, Glasgow, United Kingdom; and Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Odense, Denmark (T.U.).
authors.forename: Brian D
authors.initials: BD
authors.lastname: Hudson
authors.affiliation: Medical Research Council Toxicology Unit, University of Leicester, Leicester, United Kingdom (R.P., A.J.B., A.B.T.); Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology (E.A.-C., B.D.H., G.M.), and Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary, and Life Sciences (A.M.M.), University of Glasgow, Glasgow, United Kingdom; and Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Odense, Denmark (T.U.).
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Butcher
authors.affiliation: Medical Research Council Toxicology Unit, University of Leicester, Leicester, United Kingdom (R.P., A.J.B., A.B.T.); Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology (E.A.-C., B.D.H., G.M.), and Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary, and Life Sciences (A.M.M.), University of Glasgow, Glasgow, United Kingdom; and Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Odense, Denmark (T.U.).
authors.forename: Trond
authors.initials: T
authors.lastname: Ulven
authors.affiliation: Medical Research Council Toxicology Unit, University of Leicester, Leicester, United Kingdom (R.P., A.J.B., A.B.T.); Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology (E.A.-C., B.D.H., G.M.), and Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary, and Life Sciences (A.M.M.), University of Glasgow, Glasgow, United Kingdom; and Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Odense, Denmark (T.U.).
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Miller
authors.affiliation: Medical Research Council Toxicology Unit, University of Leicester, Leicester, United Kingdom (R.P., A.J.B., A.B.T.); Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology (E.A.-C., B.D.H., G.M.), and Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary, and Life Sciences (A.M.M.), University of Glasgow, Glasgow, United Kingdom; and Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Odense, Denmark (T.U.) graeme.milligan@glasgow.ac.uk tba@leicester.ac.uk.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Tobin
authors.affiliation: Medical Research Council Toxicology Unit, University of Leicester, Leicester, United Kingdom (R.P., A.J.B., A.B.T.); Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology (E.A.-C., B.D.H., G.M.), and Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary, and Life Sciences (A.M.M.), University of Glasgow, Glasgow, United Kingdom; and Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Odense, Denmark (T.U.) graeme.milligan@glasgow.ac.uk tba@leicester.ac.uk.
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D003412:Cricetulus; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000037:antagonists & inhibitors / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D049990:Membrane Transport Modulators / Q000494:pharmacology; D051379:Mice; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000819:agonists / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*
chemical_list: D019390:Arrestins; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; C498007:FFAR4 protein, mouse; D008164:Luminescent Proteins; D049990:Membrane Transport Modulators; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; C082333:arrestin3; C054562:yellow fluorescent protein, Bacteria; D013912:Threonine; D012694:Serine; C514862:Akt1 protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D043802:GTP-Binding Protein alpha Subunits, Gq-G11