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Molecular Pharmacology

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Research ArticleArticle

Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein–Coupled Receptor 120

Rudi Prihandoko, Elisa Alvarez-Curto, Brian D. Hudson, Adrian J. Butcher, Trond Ulven, Ashley M. Miller, Andrew B. Tobin and Graeme Milligan
Molecular Pharmacology May 2016, 89 (5) 505-520; DOI: https://doi.org/10.1124/mol.115.101949
Rudi Prihandoko
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.)
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Elisa Alvarez-Curto
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.)
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Brian D. Hudson
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.)
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Adrian J. Butcher
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.)
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Trond Ulven
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.)
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Ashley M. Miller
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.)
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Andrew B. Tobin
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.)
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Graeme Milligan
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.)
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Abstract

It is established that long-chain free fatty acids including ω-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 (Thr347, Thr349, and Ser350) and cluster 2 (Ser357 and Ser361). 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/11 proteins 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.

Footnotes

    • Received October 12, 2015.
    • Accepted February 11, 2016.
  • R.P. and E.A.-C. contributed equally to this work.

  • This research was supported by the Research Councils UK Biotechnology and Biosciences Research Council [Grants BB/K019864/1 and BB/K019856/1] and the Research Councils UK Medical Research Council Toxicology Unit [5TR60].

  • dx.doi.org/10.1124/mol.115.101949.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

  • Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 89 (5)
Molecular Pharmacology
Vol. 89, Issue 5
1 May 2016
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Research ArticleArticle

Phosphorylation Profile of mFFA4 Determines Signaling

Rudi Prihandoko, Elisa Alvarez-Curto, Brian D. Hudson, Adrian J. Butcher, Trond Ulven, Ashley M. Miller, Andrew B. Tobin and Graeme Milligan
Molecular Pharmacology May 1, 2016, 89 (5) 505-520; DOI: https://doi.org/10.1124/mol.115.101949

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Research ArticleArticle

Phosphorylation Profile of mFFA4 Determines Signaling

Rudi Prihandoko, Elisa Alvarez-Curto, Brian D. Hudson, Adrian J. Butcher, Trond Ulven, Ashley M. Miller, Andrew B. Tobin and Graeme Milligan
Molecular Pharmacology May 1, 2016, 89 (5) 505-520; DOI: https://doi.org/10.1124/mol.115.101949
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