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 including v-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 G q / 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.

Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling

“…We recently defined the sites of agonist-regulated phosphorylation within the C-terminal tail of both mouse (m)FFA4 and human (h)FFA4 and defined that conversion of these serine and threonine residues to alanines produces phosphorylation-deficient (PD) forms of the receptor orthologs (21, 22). We also recently proposed that detection of agonist-regulated GPCR phosphorylation using phospho-specific antibodies could be used as a biomarker for receptor activation (24).…”

“…We also recently proposed that detection of agonist-regulated GPCR phosphorylation using phospho-specific antibodies could be used as a biomarker for receptor activation (24). Here we used phospho-specific antibodies against the agonist-regulated phosphorylation sites Thr 347 and Ser 350 (21, 22) as a marker for FFA4 activation in genome-edited HEK293 cells. After stable expression of mFFA4-eYFP in each of parental HEK293 cells and the Gα q /Gα 11 or arrestin2/3 genome-edited cell lines and selection of individual clones, activation of mFFA4 by the agonist TUG-891 (25–27) was produced no-matter the genetic status of the cells (parental or genome-edited) (Fig.…”

“…Each of these lines was then further transfected to stably express either wild type FFA4 or a form of this receptor that cannot be phosphorylated in response to an agonist ligand because each of the residues in the C-terminal tail that becomes phosphorylated in the wild type receptor has been mutated to alanine (21, 22). We show that either restricting interaction of FFA4 with arrestins via this mutational strategy or eliminating expression of the arrestins results in prolongation of Ca 2+ signaling via FFA4, whereas we also show that arrestins do not contribute directly to FFA4-mediated ERK1/2 MAP kinase phosphorylation/activation in HEK293 cells.…”

The Multiple Waves of Cannabinoid 1 Receptor Signaling

“…These changes in receptor conformation are detected by G protein-coupled receptor kinases (GRKs), resulting in specific phosphorylation patterns at receptor intracellular domains. Quantitative mass spectrometry approaches, combined with phosphospecific antibodies, have shown that ligand-induced receptor phosphorylation is tissue and ligand specific and can be associated with specific signaling cascades; this supports a phosphorylation barcode hypothesis (Butcher et al, 2011;Liggett, 2011;Nobles et al, 2011;Prihandoko et al, 2016). Receptor phosphorylation is recognized by b-arrestins, which are recruited to the plasma membrane and sterically hinder G protein association while initiating b-arrestin-mediated internalization and signaling (Nobles et al, 2011;Liggett, 2011).…”

Cannabinoid Receptors in the Central Nervous System: Their Signaling and Roles in Disease

“…These bar-codes are finely tuned and define which signaling cascades are activated, thus opening up a spectrum of possibilities frequently defined as functional selectivity or ligand bias (Liggett, 2011; Nobles et al, 2011; Prihandoko et al, 2016). However, careful consideration must be taken when interpreting results obtained from heterologous systems, particularly when signaling can be significantly affected (biased) by the different levels of protein expression across different cell types (Bosier et al, 2010; Atwood et al, 2011; Straiker et al, 2012).…”