MRGPRX4 is a bile acid receptor for human cholestatic itch
- Peking University School of Life Sciences, China
- PKU-IDG/McGovern Institute for Brain Research, China
- Peking-Tsinghua Center for Life Sciences, China
- University of Pennsylvania, United States
- Peking University, China
- Peking University First Hospital, Peking University, China
- Peking University Third Hospital, Peking University, China
- Chinese Academy of Sciences, China
- Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, China
- Peking University First Hospital, China
- Chinese Institute for Brain Research, China
Figures
Figure 1 with 1 supplement
MRGPRX4 is activated by bile extract.
(a) Flow chart for the strategy used to identify GPCRs enriched in human DRG. Transcriptome analysis of DRG and other tissues (trigeminal ganglia, brain, colon, liver, lung, skeletal muscle, and …
Figure 1—figure supplement 1
Construct design and surface expression of candidate GPCRs in HEK293T cells.
(a) Map of the generic GPCR expression vector. The 3’ and 5’ terminal repeats (TR) are recognized by the PiggyBac transposase. Myc, Myc tag; PuroR, puromycin-resistance gene. (b) Plasma membrane …
Figure 2 with 1 supplement
Identification of the active components in bile extract that activate MRGPRX4.
(a) Flow chart depicting the strategy for isolating and identifying candidate MRGPRX4 ligands in bovine bile extract. F1 through F6 indicate the six fractions used in subsequent experiments. (b) …
Figure 2—figure supplement 1
1H-NMR analysis of bile acids in fractions F1, F2, F3, F4, and F6.
The hydrogen chemical shift of CA, CDCA, DCA, and LCA at carbon 3, 7, 12 were determined by 1H-NMR. Note that the active fractions (F2 through F4) contained the characteristic hydrogen peaks …
Figure 3 with 1 supplement
Functional characterization and molecular profiling of bile acids as ligands for MRGPRX4.
(a–c) Dose-dependent activation of MRGPRX4 by various bile acids and their derivatives. MRGPRX4 activation was measured using the TGFα shedding assay (a) or the FLIPR assay (b) see Materials and meth…
Figure 3—figure supplement 1
Human MRGPRX4, but not human MRGPRX1-3 or mouse and rat Mrgpr family members, are activated by bile acids.
(a) Phylogenetic analysis of mouse (Mm, green), rat (Rn, blue), rhesus monkey (Rh, black), and human (Hs, red) Mas-related GPCR (Mrg) family members. Amino acid sequence similarity compared to Hs. …
Figure 4 with 1 supplement
MRGPRX4 is expressed in a subset of hDRG neurons.
(a–d) Representative DRG sections showing in situ hybridization (ISH, (a) and immunohistochemistry (IHC, (b) for MRGPRX4; the summary data are shown in (c); n = 2234 and 2735 neurons for ISH and …
Figure 4—figure supplement 1
The anti-MRGPRX4 antibody has high specificity.
HEK293T cells were transiently transfected with MRGPRX1, MRGPRX2, MRGPRX3, or MRGPRX4. The anti-MRGPRX4 antibody (Abcam, ab120808, 1:200 dilution) specifically labeled MRGPRX4-expressing HEK293T …
Figure 5 with 2 supplements
MRGPRX4 mediates bile acids induced activation of DRG neurons.
(a) Top, cultured rat DRG neurons were transfected with the pPiggyBac-CAG-MRGPRX4-P2A-mCherry plasmid by electroporation. The neurons circled by dashed lines are an MRGPRX4-positive neuron (neuron …
Figure 5—figure supplement 1
Expressing MRGPRX4 in cultured rat DRG neurons renders the cells responsive to bile acids.
(a) Non-transfected cultured rat DRG neurons. A representative neuron is circled by the dash line. Scale bar, 50 μm. (b) Representative traces from the cells indicated in (a). DCA and CA: 10 μM; …
Figure 5—figure supplement 2
Cultured human DRG neurons respond to various chemicals.
(a) Full size pseudo-color images of chemical-induced signals from cultured human embryo DRG neurons (Figure 5g). (b1–b3) Representative bright-field and fluorescence images of cultured human adult …
Figure 6
Bile acids and MRGPRX4 specific agonist induce histamine-independent itch in human.
(a1–a2) Itch evoked by a double-blind intradermal injection of DCA and nateglinide (Nat) in human subjects. (25 μl for each injection) (a1) Time courses of the perceived itch intensity (n = 18–32). …
Figure 7 with 1 supplement
TGR5 does not serve as an itch receptor in human.
(a1–a2) Intradermal injection of a non-bile acid TGR5 agonist compound 15 (C15) does not induce itch in human. (a1) Time courses of the perceived intensity of itch evoked by DCA and vehicle are …
Figure 7—figure supplement 1
Expression of TGR5 in mouse and monkey DRG.
(a) Phylogenetic analysis of mouse (Mm.), rat (Rn.), rhesus monkey (Rh,) and human (Hs,) TGR5. Amino acid sequence similarity compared to Hs. TGR5 is shown in the parenthesis. (b) The HEK293T cells …
Figure 8 with 1 supplement
Elevated bile acids are correlated with the occurrence of itch among patients with liver disease and are sufficient to activate MRGPRX4.
(a–b) Summary of individual bile acid levels (a) and total bile acid levels (b), the sum of the 12 bile acids shown in a) in itchy patients with liver diseases (Liver_itch, n = 27), non-itchy …
Figure 8—figure supplement 1
Quantification of bile acids in human plasma.
(a) Standard curve of 12 bile acids quantified by HPLC-MS/MS. All the 12 bile acids show good linear correlation between the MS response and the concentration (0.1–1 μM). (b) Quantification results …
Figure 9
Bilirubin potentiates the activation of MRGPRX4 by bile acids and may contribute to cholestatic itch.
(a) Comparison of the activation of MRGPRX4 by DCA, bilirubin and taurine conjugated bilirubin. Taurine conjugated bilirubin was used in order to mimic the direct bilirubin under human physiological …
Figure 10
Proposed model depicting the mechanism underlying itch in patients with liver diseases.
In itchy patients, accumulated bile acids reach the skin via the circulatory system, where they activate nerve fibers of a subset of MRGPRX4-expressing DRG neurons. Bilirubin, as a partial agonist, …
Author response image 1
Representative large-scale RNAscope images of human DRG.
Each fluorescent dot indicated by an arrowhead represents one single mRNA transcript. The large fluorescent dots indicated by arrows are non-specific background (positive for all three channels). …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (H. sapiens) | MRGPRX4 | hORFeome Database | Genbank Accession: KJ900052 | |
| Gene (H. sapiens) | TGR5 | hORFeome Database | Genbank Accession: KJ895655 | |
| Cell line (Homo-sapiens) | HEK stable cell line for TGFα shedding assay | PMID: 22983457 | Self-generated according to reference paper | |
| Chemical compound, drug | Screen Quest Fluo-8 No Wash Calcium Assay Kit | AAT Bioquest | Cat. #: 36316 | |
| Chemical compound, drug | Fluo-8, AM | AAT Bioquest | Cat. #: 21083 | |
| Chemical compound, drug | Deoxycholic acid | Sigma-Aldrich | Cat. #: D2510 | |
| Chemical compound, drug | U73122 | Selleck | Cat. #: S8011 | |
| Chemical compound, drug | Nateglinide | J and K Scientific | Cat. #: 586681 | |
| Chemical compound, drug | Bilirubin ditaurate | J and K Scientific | Cat. #: F-H130 | |
| Chemical compound, drug | Compound 15 | custom- synthesized | Verified by HPLC and mass spectrum | |
| Chemical compound, drug | Histamine | Sigma-Aldrich | Cat. #: H7250 | |
| Antibody | Rabbit polyclonal anti-hMRGPRX4 | Abcam | Cat #: ab120808 | IHC (1:200) |
| Antibody | Rabbit polyclonal anti-hTGR5 | Thermofisher | Cat #: PA5-27076 RRID:AB_2544552 | IHC (1:200-1:500) |
| Antibody | Mouse monoclonal anti-NeuN (clone A60) | Sigma-Aldrich | Cat. #: MAB377 RRID:AB_2298772 | IHC (1:1000) |
| Antibody | Rabbit polyclonal anti-c-Myc | Sigma-Aldrich | Cat. #: C3956 RRID: AB_439680 | IHC (1:200) FACS (1:50) |
| Antibody | Sheep polyclonal anti-Digoxigenin-AP | Roche | Cat. #: 11093274910 RRID:AB_2734716 | ISH (1:1000) |
| Commercial assay or kit | RNAscope Fluorescent Multiplex Assay | Advanced Cell Diagnostics | Cat. #: 320293 UM | |
| Commercial assay or kit | P3 Primary Cell 4D-NucleofectorTM X Kit L | Lonza | Cat. # V4XP-3012 | |
| Recombinant DNA reagent | pPiggyBac-MRGPRX4-P2A-mCherry (Plasmid) | This paper | See in Figure 1—figure supplement 1a | |
| Recombinant DNA reagent | pPiggyBac-TGR5-P2A-mCherry (Plasmid) | This paper | See in Figure 1—figure supplement 1a | |
| Other | Doxepin hydrochloride cream | Chongqing Huapont Pharm. Co. | ||
| Other | Cold cream | Eau Thermale Avène |
Additional files
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Supplementary file 1
Genes that are highly expressed in human DRG.
- https://doi.org/10.7554/eLife.48431.020
-
Supplementary file 2
GPCRs expression profiling in human DRG.
Red labeled genes are candidate GPCRs that are highly expressed in human DRG. The blue labeled gene is TGR5.
- https://doi.org/10.7554/eLife.48431.021
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Transparent reporting form
- https://doi.org/10.7554/eLife.48431.022
