A molecular mechanism for sequential activation of a G Protein-coupled receptor

Grundmann, M., Tikhonova, I. G., Hudson, B. D. , Smith, N. J., Mohr, K., Ulven, T., Milligan, G. , Kenakin, T. and Kostenis, E. (2016) A molecular mechanism for sequential activation of a G Protein-coupled receptor. Cell Chemical Biology, 23(3), pp. 392-403. (doi: 10.1016/j.chembiol.2016.02.014) (PMID:26991104)

119571.pdf - Accepted Version



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.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Hudson, Dr Brian and Milligan, Professor Graeme
Authors: Grundmann, M., Tikhonova, I. G., Hudson, B. D., Smith, N. J., Mohr, K., Ulven, T., Milligan, G., Kenakin, T., and Kostenis, E.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Cell Chemical Biology
Published Online:17 March 2016
Copyright Holders:Copyright © 2016 Elsevier
First Published:First published in Cell Chemical Biology 23(3):392-403
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher

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