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 sorbic acid or MOMBA. Composite outcomes of a series of independent experiments are combined. Fragmentation tables associated with phosphorylated peptides are shown. Phosphorylated residues are highlighted in red.

Characteristics of putative pSer296 /pSer297 and pThr306 /pThr310 hFFA2- antisera and the effect of potential phospho-acceptor site mutations on agonistinduced arrestin-3 interactions

The primary amino acid sequence of hFFA2 is shown (A). Residues altered to generate the DREADD variant are in red (Cys141 Gly, His242 Gln). 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. Each of the DREADD-PD variants, except hFFA2-DREADD-PD2 (ns), were less effective in promoting interactions in response to MOMBA (** p < 0.01, **** p < 0.0001). 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 (*** p < 0.001). Significance in C and D were assessed by one-way ANOVA followed by Dunnett’s multiple comparisons test. E. The effect of compound 101 on detection of hFFA2- DREADD-eYFP by each of pSer296 /pSer297, pThr306 /Thr310 and anti-GFP antisera is shown. Data are representative (B, E) or show means +/- SEM (C, D) of at least 3 independent experiments.

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-DREADDeYFP. 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.

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.

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. ROR©T 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).

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.

Propionate regulates phosphorylation of hFFA2-eYFP: In vitro studies

Flp-In T-REx 293 cells habouring 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-pSer 296 /pSer 297 hFFA2, anti-pThr 306 /pThr 310 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-pThr 306 /pThr 310 hFFA2. C., D. Cells were doxycycline induced (+ dox) or not (-dox) and prepared for immunocytochemistry after treatment with C3 or vehicle and exposed to antipThr 306 /pThr 310 hFFA2 (C) or anti-pSer 296 /pSer 297 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.

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-pThr 306 /pThr 310 (A) or anti-pSer 296 /pSer 297 (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-pThr 306 /pThr 310 (C, centre) or anti-pSer 296 /pSer 297 (C, right). hFFA2-HA was detected as a broad smear of protein(s) with Mr centred close to 55 kDa. D. Quantification of pThr 306 /pThr 310 (left) and pSer 296 /pSer 297 immunoblots (right) phosphorylation in experiments using tissue from three different mice (means +/- S.E.M.), * p < 0.05, ns: not significant.

hFFA2-DREADD-HA becomes phosphorylated at Thr 306 /Thr 310 in immune cells within Peyer’s patches

Experiments akin to those of Figure 5F were conducted using the pThr 306 /pThr 310 hFFA2 antiserum. The figure illustrates MOMBA-induced phosphorylation of pThr 306 /pThr 310 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.

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 SDSPAGE 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.

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.