Distinct phosphorylation clusters determines the signalling outcome of the free fatty acid receptor FFA4/GPR120

Prihandoko, R., Alvarez-Curto, E. , Hudson, B. D. , Butcher, A. J., Ulven, T., Miller, A. M., Tobin, A. B. and Milligan, G. (2016) Distinct phosphorylation clusters determines the signalling outcome of the free fatty acid receptor FFA4/GPR120. Molecular Pharmacology, 89(5), pp. 505-520. (doi:10.1124/mol.115.101949) (PMID:26873857)

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Abstract

It is established that long-chain free fatty acids including ω-3 fatty acids mediate an array of biological responses through members of the free fatty acid receptor family, which includes FFA4. However, the signalling mechanisms and modes of regulation of this receptor class remain unclear. Here we employ 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 phospho-acceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to ERK1/2 activation. Rather an inhibitor of Gq/11 proteins completely prevented receptor signalling to ERK1/2. In contrast, the recruitment of arrestin 3, receptor internalization and activation of Akt were regulated by mFFA4 phosphorylation. The analysis of mFFA4 phosphorylation-dependent signalling was extended further by selective mutations of the phospho-acceptor 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 phospho-acceptor sites within cluster 1 had no effect on receptor internalization and a less extensive effect on arrestin 3 recruitment, but significantly uncoupled the receptor from Akt activation. These unique observations define differential effects on signalling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signalling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode) at the C-terminus of the receptor.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Milligan, Professor Graeme and Miller, Dr Ashley and Alvarez-Curto, Dr Elisa and Hudson, Dr Brian
Authors: Prihandoko, R., Alvarez-Curto, E., Hudson, B. D., Butcher, A. J., Ulven, T., Miller, A. M., Tobin, A. B., and Milligan, G.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Molecular Pharmacology
Publisher:American Society for Pharmacology and Experimental Therapeutics
ISSN:0026-895X
ISSN (Online):1521-0111

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