Chemogenetic Approaches to Explore the Functions of Free Fatty Acid Receptor 2

Milligan, Graeme; Barki, Natasja; Tobin, Andrew B.

doi:10.1016/j.tips.2020.12.003

Cited by 12 publications

(11 citation statements)

References 70 publications

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“…To assess physiological roles of FFA2 in mouse tissues without potential confounding effects of either activation of the related SCFA-receptor FFA3 or non-receptor mediated effects of SCFAs we recently developed a hFFA2-DREADD knock-in transgenic mouse line (29). Here, mouse FFA2 is replaced by hFFA2-DREADD with, in addition, an appended C- terminal anti-HA epitope tag sequence to allow effective identification of cells expressing the receptor construct (20, 28).…”

Section: Resultsmentioning

confidence: 99%

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Phosphorylation bar-coding of Free Fatty Acid receptor 2 is generated in a tissue-specific manner

Barki,

Jenkins,

Marsango

et al. 2023

Preprint

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Free Fatty Acid receptor 2 (FFA2) is activated by short-chain fatty acids and expressed widely, including in white adipocytes and various immune and enteroendocrine cells. Using both wild type human FFA2 and a Designer Receptor Exclusively Activated by Designer Drugs (DREADD) variant we explored the activation and phosphorylation profile of the receptor, both in heterologous cell lines and in tissues from transgenic knock-in mouse lines expressing either human FFA2 or the FFA2-DREADD. FFA2 phospho-site specific antisera targeting either pSer296/pSer297 or pThr306/pThr310 provided sensitive biomarkers of both constitutive and agonist-mediated phosphorylation as well as an effective means to visualise agonist-activated receptors in situ. In white adipose tissue phosphorylation of residues Ser296/Ser297 was enhanced upon agonist activation whilst Thr306/Thr310 did not become phosphorylated. By contrast, in immune cells from Peyers patches Thr306/Thr310 become phosphorylated in a strictly agonist-dependent fashion whilst in enteroendocrine cells of the colon both Ser296/Ser297 and Thr306/Thr310 were poorly phosphorylated. The concept of phosphorylation bar-coding has centred to date on the potential for different agonists to promote distinct receptor phosphorylation patterns. Here we demonstrate that this occurs for the same agonist-receptor pairing in different patho-physiologically relevant target tissues. This may underpin why a single G protein-coupled receptor can generate different functional outcomes in a tissue-specific manner.

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Section: Resultsmentioning

confidence: 99%

“…Most significantly in the context of phosphorylation bar-coding we were able to show tissue-selective phosphorylation of Thr 306 and Thr 310 . To do so we took advantage of a knockin transgenic mouse line that we have characterized extensively (20,(28)(29). In this line we replaced mouse FFA2 with hFFA2-DREADD.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation bar-coding of Free Fatty Acid receptor 2 is generated in a tissue-specific manner

Barki,

Jenkins,

Marsango

et al. 2023

Preprint

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“…The interaction and binding properties of the two FFAR2 specific antagonist (CATPB and GLPG0974) included in the study have previously been fairly well characterized/documented [10,11,13,35,36]. Although their detailed binding characteristics differ, it has been shown that both antagonists fit into, and bind specifically to the orthosteric site/binding pocket in human FFAR2 and that their respective affinity for the receptor are very similar or even identical [7,13]. Accordingly, both antagonists inhibit the response induced by natural (i.e., short chain fatty acids) as well as synthetic (i.e., small compounds such as Cmp1; [11,37] orthosteric FFAR2 agonists.…”

Section: Discussionmentioning

confidence: 99%

“…Members of this receptor family are all membrane proteins that change their basic conformation when agonists (activating ligands) bind to the so called orthosteric binding site exposed on the membrane surface of receptor-expressing cells [4,5]. Free fatty acid receptor 2 (FFAR2 [6,7]) is such a receptor expressed in neutrophils. This receptor recognizes short fatty acids (FFAs) such as acetate and propionate, metabolites produced by gut microorganisms during fermentation of dietary fibers.…”

Section: Introductionmentioning

confidence: 99%

Allosteric receptor modulation uncovers an FFAR2 antagonist as a positive orthosteric modulator/agonist in disguise

Lind

Hoffman

Forsman

et al. 2021

Preprint

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Two earlier described Free Fatty Acid Receptor 2 (FFAR2)-specific antagonists (CATPB and GLPG0974) have different receptor-interaction characteristics at the molecular/functional level. The inhibitory effect of the two antagonists, on the novel receptor-cross-talk activation signals generated by the ATP-receptor, show that both antagonists inhibit the effect of the positive allosteric FFAR2 modulators (PAMs) AZ1729 and Cmp58. No neutrophil activation was induced by AZ1729 or Cmp58 alone, but together they were co-agonistic PAMs and activated the superoxide generating NADPH-oxidase in neutrophils. This response was inhibited by CATPB but not by GLPG0974; in contrast, GLPG0974 acted as a positive modulator that increased the potency but not the efficacy of the response. At the signaling level, GLPG0974 changed the biased signaling induced by the co-agonistic PAMs, to include a rise in the cytosolic concentration of free calcium ions (Ca2+). This effect was reciprocal, i.e., GLPG0974 triggers a rise in intracellular Ca2+, demonstrating that GLPG0974 may act as an FFAR2 agonist. In summary, by studying the effects of the FFAR2 ligand GLPG0974 on neutrophils activation induced by the co-agonists AZ1729 and Cmp58, we reveal that GLPG0974 in addition to be an antagonist, displays also agonistic and positive FFAR2 modulating functions that affects the NADPH-oxidase activity and the receptor down-stream signaling induced by the two co-agonistic PAMs.

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“…To explore the role of regulated phosphorylation of FFA4, the wild type receptor was replaced with a variant in which all the hydroxy-amino acids in the C-terminal tail were replaced by alanines ( phospho-deficient, PD ). In addition, to facilitate detection of the expressed receptor protein, the HA-epitope tag sequence was placed in-frame at the C-terminus, as previously reported for the free fatty acid receptor 2 [ 138 , 139 , 140 ]. Together with the wild type and FFA4 knock-out littermates, a comprehensive understanding of the role of the phosphorylation of FFA4 may be established by studying this line.…”

Section: Figurementioning

confidence: 99%

How Arrestins and GRKs Regulate the Function of Long Chain Fatty Acid Receptors

Alharbi

Tobin

Milligan

2022

IJMS

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FFA1 and FFA4, two G protein-coupled receptors that are activated by long chain fatty acids, play crucial roles in mediating many biological functions in the body. As a result, these fatty acid receptors have gained considerable attention due to their potential to be targeted for the treatment of type-2 diabetes. However, the relative contribution of canonical G protein-mediated signalling versus the effects of agonist-induced phosphorylation and interactions with β-arrestins have yet to be fully defined. Recently, several reports have highlighted the ability of β-arrestins and GRKs to interact with and modulate different functions of both FFA1 and FFA4, suggesting that it is indeed important to consider these interactions when studying the roles of FFA1 and FFA4 in both normal physiology and in different disease settings. Here, we discuss what is currently known and show the importance of understanding fully how β-arrestins and GRKs regulate the function of long chain fatty acid receptors.

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Chemogenetic Approaches to Explore the Functions of Free Fatty Acid Receptor 2

Cited by 12 publications

References 70 publications

Phosphorylation bar-coding of Free Fatty Acid receptor 2 is generated in a tissue-specific manner

Phosphorylation bar-coding of Free Fatty Acid receptor 2 is generated in a tissue-specific manner

Allosteric receptor modulation uncovers an FFAR2 antagonist as a positive orthosteric modulator/agonist in disguise

How Arrestins and GRKs Regulate the Function of Long Chain Fatty Acid Receptors

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