Figures and data
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Mass spectrometry analysis of hFFA2-DREADD-eYFP identifies basal phosphorylation of Ser297 and agonist-promoted phosphorylation of Ser296
Mass spectrometry analysis was conducted on samples isolated from Flp-In T-REx 293 cells in which expression of hFFA2-DREADD-eYFP had been induced. Experiments were performed on vehicle and MOMBA-treated (100 μM, 5 min) cells as detailed in Experimental. LC-MS/MS identified Ser297 as being phosphorylated constitutively, and Ser296/297 as being phosphorylated by MOMBA (and also by sorbic acid, not shown). Composite outcomes of a series of independent experiments are combined. Fragmentation tables associated with phosphorylated peptides are shown. Phosphorylated residues are highlighted in red.
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Characteristics of putative pSer296/pSer297 and pThr306/pThr310 hFFA2-antisera and the effect of potential phospho-acceptor site mutations on agonist-induced arrestin-3 interactions
The primary amino acid sequence of hFFA2 is shown (A). Residues altered to generate the DREADD variant are in red (Cys141Gly, His242Gln). Phospho-deficient (PD) hFFA2-DREADD variants were generated by replacing serine 296 and serine 297 (purple, hFFA2-DREADD-PD1), threonine 306 and threonine 310 (light blue, hFFA2-DREADD-PD2), serine 324 and serine 325 (dark blue, hFFA2-DREADD-PD3) or threonine 328 and 329 (yellow, hFFA2-DREADD-PD4) with alanine. In addition, hFFA2-DREADD-PD1-4 was generated by combining all these alterations, B. The ability of putative pSer296/pSer297 and pThr306/Thr310 antisera to identify wild type and either PD1 or PD2 forms of hFFA2-DREADD with and without treatment of cells expressing the various forms with MOMBA is shown and, as a control, anti-GFP immunoblotting of equivalent samples is illustrated. C. The ability of varying concentrations of MOMBA to promote interaction of arrestin-3 with hFFA2-DREADD and each of the DREADD-PD mutants, is illustrated. D. The effect of the GRK2/3 inhibitor compound 101 on the capacity of MOMBA to promote recruitment of arrestin-3 to wild type hFFA2-DREADD is shown. Data are illustrative (B) or represent means +/-SEM (C, D) of at least 3 independent experiments.
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Agonist-induced detection of hFFA2-DREADD with putative pSer296/pSer297 and pThr306/pThr310 antisera reflects receptor activation, receptor phosphorylation and can be detected in situ
The ability of the pSer296/pSer297, pThr306/pThr310 hFFA2 and, as a control GFP, antisera to identify hFFA2-DREADD-eYFP after induction to express the receptor construct and then treatment of cells with vehicle, MOMBA, sorbic acid (each 100 µM) or propionate (C3) (2 mM) is shown. In the ‘-dox’ lanes receptor expression was not induced. B. as in A. except that after cell treatment with vehicle or MOMBA, immune-enriched samples were treated with Lambda Protein Phosphatase (LPP) or, rather than treatment with MOMBA, cells were treated with a combination of MOMBA and the hFFA2 inverse agonist CATPB (10 µM, 30 min pre-treatment). C, D. Cells harboring hFFA2-DREADD-eYFP and grown on glass coverslips were either untreated (–dox) or induced to express hFFA2-DREADD-eYFP. The induced cell samples were then exposed to vehicle, MOMBA (100 µM) or a combination of MOMBA (100 µM) and CATPB (10 µM) for 5 min. Fixed cells were then treated with anti-pThr306/pThr310 (C) or anti-pSer296/pSer297 (D) (red, Alexa Fluor 647) or imaged to detect eYFP (green). DAPI was added to detect DNA and highlight cell nuclei (blue). Scale bar = 20 μm.
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In white adipose tissue residues Ser296/Ser297 of hFFA2-DREADD-HA but not Thr306/Thr310 become phosphorylated in response to MOMBA
White adipose tissue dissected from hFFA2-DREADD-HA and CRE-MINUS mice was treated with either vehicle, 100 µM MOMBA or 100 µM MOMBA + 10 µM CATPB A. Lysates were prepared and solubilized. hFFA2-DREADD-HA was immunoprecipitated using an anti-HA monoclonal antibody and following SDS-PAGE immunoblotted to detect HA, non-phosphorylated hFFA2-DREADD-HA, and pSer296/pSer297 or pThr306/pThr310 hFFA2-DREADD-HA. A representative experiment is shown. B. Quantification of pSer296/pSer297 (left) and pThr306/pThr310 immunoblots (right) phosphorylation (means +/-S.E.M.) in experiments using tissue from different mice, * p < 0.05, ns: not significant. C. Tissue samples from hFFA2-DREADD-HA (top panel) and CRE-MINUS (bottom panel) mice that were treated with MOMBA were immunostained to detect pSer296/pSer297 (left panels), pThr306/pThr310 (right panels) and counterstained with DAPI (blue). Scale bars = 20 µm. D. Comparison of pSer296/pSer297 staining of samples from hFFA2-DREADD-HA expressing mice vehicle treated (top panels) or treated with MOMBA (bottom panels) (scale bar = 50 µm). E, F. Tissue sections from hFFA2-DREADD-HA-expressing mice immunostained with pSer296/pSer297 (green) and anti-HA (red) (E) to detect the receptor expression or F with anti-perilipin-1 (red) to identify adipocytes. Merged images are shown to the right. Scale bars = 20 µm.
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hFFA2-DREADD-HA becomes phosphorylated at Thr306/Thr310 in addition to Ser296/Ser297 in immune cells from Peyer’s patches
Peyer’s patches isolated from hFFA2-DREADD-HA expressing mice were immunostained with anti-HA (red) to detect receptor expression. Images were acquired with ×20 (left panel) and ×63 (right panel) objectives (scale bar = 200 µm) (A). Tissue sections were counterstained with B. anti-CD11c as a marker of dendritic cells, monocytes and/or macrophages or, C. RORγT to detect type-III innate lymphoid cells (scale bar = 20 µm). Isolated Peyer’s patches and mesenteric lymph nodes from CRE-MINUS and hFFA2-DREADD-HA mice were exposed to either vehicle, 100 µM MOMBA or 100 µM MOMBA + 10 µM CATPB. D. Following lysate preparation, immunoprecipitation and SDS-PAGE samples were probed to detect HA, pThr306/pThr310 or pSer296/pSer297. E. Quantification of pThr306/pThr310 (left) and pSer296/pSer297 immunoblots (right) hFFA2-DREADD-HA (means +/-S.E.M.), *p < 0.05, **p < 0.01. F. Treated tissue sections were also used in immunohistochemical studies, employing either pThr306/pThr310 (left panels) or pSer296/pSer297 (right panels) (scale bars = 100 µm).
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MOMBA promotes limited phosphorylation of both Ser296/Ser297 and Thr306/Thr310 in hFFA2-DREADD-HA in lower gut enteroendocrine cells
A. B., Colonic tissue isolated from hFFA2-DREADD-HA (top panels) or CRE-MINUS (bottom panels) mice treated with either vehicle (A) or 100 µM MOMBA (B). Following fixation tissue sections were immunostained with pThr306/pThr310 and counterstained with DAPI. (Scale bar = 100 µm). In the merged images, the box is expanded in the right-hand panels. C. Lysates prepared from tissue samples treated as noted were analysed by probing immunoblots with anti-HA, anti-pThr306/pThr310 or anti-pSer296/pSer297. Representative examples are shown.
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Propionate regulates phosphorylation of hFFA2-eYFP: In vitro studies
Flp-In T-REx 293 cells haboring hFFA2-eYFP were induced to express the receptor construct (+ dox) or not (–dox) and the induced cells were then treated with propionate (C3, 2 mM, 5 min) or vehicle. A. Cell lysates were resolved by SDS-PAGE and then immunoblotted with anti-pSer296/pSer297 hFFA2, anti-pThr306/pThr310 hFFA2, or anti-GFP. B. Cells induced to express hFFA2-eYFP were treated with C3 (2 mM, 5 min) or vehicle. Where noted cells were pre-treated with the hFFA2 antagonist CATPB (10 μM, 20 min before agonist addition). Lysates were then prepared and, where indicated, treated with Lambda Protein Phosphatase (LPP). Following SDS-PAGE the samples were immunoblotted with anti-pThr306/pThr310 hFFA2. C., D. Cells were doxycycline induced (+ dox) or not (-dox) and prepared for immunocytochemistry after treatment with C3 or vehicle and exposed to anti-pThr306/pThr310 hFFA2 (C) or anti-pSer296/pSer297 hFFA2 (D) (red) whilst direct imaging detected the presence of hFFA2-eYFP (green). Merged images (right hand panels) were also stained with DAPI (blue) to identify cell nuclei. Scale bars = 20 µm.
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C3-induces phosphorylation of both pSer296/pSer297 and pThr306/pThr310 in Peyer’s patches from hFFA2-HA expressing mice
Isolated Peyer’s patches and mesenteric lymph nodes from hFFA2-HA and the corresponding CRE-MINUS mice were exposed to either vehicle, or 10 mM C3 for 20 minutes. Tissue sections were used in immunohistochemical studies, employing either anti-pThr306/pThr310 (A) or anti-pSer296/pSer297 (B) (scale bars = 100 µm). C. Lysates from Peyer’s patches isolated from hFFA2-HA expressing mice, or the corresponding CRE-MINUS mice, that had been treated with vehicle, C3 (10 mM, 20 min), or C3 + CATPB (10 μM, 30 minutes before agonist) were immunoprecipitated with anti-HA as for the hFFA2-DREADD-HA expressing mice in Figure 5. Subsequent to SDS-PAGE samples such were probed to detect HA (C, left), anti-pThr306/pThr310 (C, centre) or anti-pSer296/pSer297(C, right). hFFA2-HA was detected as a broad smear of protein(s) with Mr centred close to 55 kDa. D. Quantification of pThr306/pThr310 (left) and pSer296/pSer297 immunoblots (right) phosphorylation in experiments using tissue from three different mice (means +/-S.E.M.), * p < 0.05, ns: not significant.
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hFFA2-DREADD-HA becomes phosphorylated at Thr306/Thr310 in immune cells within Peyer’s patches
Experiments akin to those of Figure 5F were conducted using the pThr306/pThr310 hFFA2 antiserum. The figure illustrates MOMBA-induced phosphorylation of pThr306/pThr310 in immune cells within Peyer’s patches of hFFA2-DREADD-HA expressing mice. Peyer’s patches were either treated with vehicle (Left panel) or MOMBA (100 µM) (Right panel). Each lymphoid nodule has been expanded to show detailed phosphorylation inside each nodule.
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Tissues of transgenic mice express similar levels of hFFA2-HA and hFFA2-DREADD-HA
White adipose (WAT) (left panel) and colonic epithelial (right panel) tissue was isolated from CRE-MINUS and both hFFA2-DREADD-HA and hFFA2-HA expressing transgenic mice. Anti-HA immunoprecipitations were then performed, and samples resolved by SDS-PAGE followed by immunoblotting with anti-HA. For both tissues similar levels and patterns of the receptor proteins were detected from the hFFA2-DREADD-HA and hFFA2-HA expressing mice, whilst these were absent in tissue from CRE-MINUS animals. A representative experiment of 3 is shown.
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hFFA2-DREADD-HA is present as multiple differentially N-glycosylated species in adipose tissue, immune, and colonic epithelial cells
HA-immunoprecipitated samples from white adipose tissue (WAT), colonic epithelium and Peyer’s patches and mesenteric lymph nodes (PP + MLNs) of hFFA2-DREADD-HA expressing mice were untreated (A) or treated with N-glycosidase F to remove N-linked carbohydrate (B). They were then resolved by SDS-PAGE and immunoblotted with anti-HA. A representative experiment of 3 is shown.