Gq activity- and β-arrestin-1 scaffolding-mediated ADGRG2/CFTR coupling are required for male fertility
- Shandong University School of Medicine, China
- Binzhou Medical University, China
- School of Medicine, Duke University, United States
- School of Basic Medical Sciences, Peking University, China
- Sungkyunkwan University, Korea
- National Health and Family Planning Commission, China
- Shandong University, China
- Shandong University School of Life Sciences, China
- Institute of Biomedical Sciences, East China Normal University, China
- Texas A&M University Health Science Center, United States
Figures
Figure 1 with 2 supplements
The expression of G protein subtypes in the efferent ductules and ADGRG2 promoter-labeled non-ciliated cells.
(A) qRT-PCR analysis of mRNA transcription profiles of G proteins in brain tissues and the efferent ductules of WT (n = 3) male mice. Expression levels were normalized to GAPDH levels. *p<0.05, **p<0…
Figure 1—figure supplement 1
ADGRG2 is specifically expressed in non-ciliated cells.
(A) Control experiments: Direct immunofluorescence staining of secondary antibodies used in the manuscript (including donkey anti-sheep, red fluorescence; and donkey anti-rabbit, green fluorescence) …
Figure 1—figure supplement 2
The construction of the mouse ADGRG2-promoter-RFP used in the labeling of ADGRG2-expressed cells.
(A–B) Schematic representation of the construction of the mouse ADGRG2-promoter-RFP used in the labeling of ADGRG2 expressed cells in the epididymal efferent duct epithelium. Sub-cloning strategy of …
Figure 2 with 2 supplements
Gq activity is required for fluid reabsorption.
(A) Images of cultured ligated efferent ductules derived from WT male mice, Adgrg2-/Y mice and Gnaq+/- male mice. Ductule segments were selected by examination of the ciliary beat, which is a marker …
Figure 2—figure supplement 1
The ADGRG2 protein knockout strategy, PCR strategy and western blot results of Adgrg2-/Y and Gnaq+/- mice.
(A) Schematic representation of the ADGRG2 knockout strategy for the Adgrg2-/Y mice. 2 bp nucleotides were removed and 10 bp nucleotides were inserted in the first exon of the ADGRG2 gene in the …
Figure 2—figure supplement 2
Effects of Forskolin and IBMX on the diameters of ligated efferent ductules derived from WT or Adgrg2-/Y mice.
(A) Effects of forskolin (10 μM), an adenylyl cyclase (AC) activator, on the diameters of ligated efferent ducts; WT(n = 11) or Adgrg2-/Y (n = 10). (B) Effects of IBMX(100 μM), a non-specific …
Figure 3
Gq expression is required for sperm transportation and male fertility.
(A) Representative hematoxylin and eosin staining of WT, Adgrg2-/Y or Gnaq+/- mice. Scale bars, 200 μm. (B–D) Corresponding bar graphs demonstrating the accumulation of spermatozoa according to the …
Figure 4 with 1 supplement
Inhibition of CFTR activity in the efferent ductules pheno-copied the activity in Adgrg2-/Y mice.
(A) qRT-PCR analysis of the mRNA transcription profiles of potential osmotic drivers including selective ion channels and transporters in ADGRG2 promoter-labeled cells, non-ADGRG2 promoter-labeled …
Figure 4—figure supplement 1
Expression and functional analysis of potential osmotic drivers in efferent ductules.
(A) Quantitative RT-PCR (qRT-PCR) analysis of mRNA transcription profiles of potential osmotic drivers including selective ion channels and transporters in efferent ductules, brain and liver of …
Figure 5 with 4 supplements
Functional coupling and co-localization of CFTR and ADGRG2 on the apical membrane in the efferent ductules.
(A) Intracellular pH (pHi) of the ligated efferent ductules from WT (n = 9) mice and Adgrg2-/Y (n = 9) mice were measured by carboxy-SNARF (5 μM), with or without incubation with the CFTR inhibitor …
Figure 5—figure supplement 1
Representative agrose gel for the reverse transcription PCR analysis of CFTR mRNA level in efferent ductules of WT or Adgrg2-/Y mice.
The upper band (220 bp PCR product) in each lane represents CFTR, whereas the lower band (100 bp product) represents GAPDH (This figure was related to Figure 5B).
Figure 5—figure supplement 2
pH homeostasis in the efferent ductules was impaired in Adgrg2-/Y mice.
(A) The relationship between R value (fluorescence emission intensity at 635nm/fluorescence emission intensity at 590 nm) and pHi for the indicator 5'(and …
Figure 5—figure supplement 3
Immunostaining experiments for CFTR location in efferent ductules.
(A) Co-Immunofluorescence staining of ADGRG2 (red fluorescence) and ANO1(green fluorescence) in WT male mice efferent ductules. Scale bars, 50 μm. (B) Immunofluorescent staining of CFTR (sc-8909, …
Figure 5—figure supplement 4
Bar graph representation and statistical analyses of Figure 5H.
***p<0.001, Adgrg2-/Y lysates or IP protein were compared with WT lysates or IP protein respectively. n.s., no significant difference.
Figure 6
The whole-cell Cl- current recording of ADGRG2 promoter-labeled efferent ductule cells.
(A) Time course of whole-cell Cl- current (IADGRG2-ED) at +100 and −100 mV in ADGRG2 promoter-labeled efferent ductule cells derived from Adgrg2-/Y mice or their littermates. An ‘a’ or ‘d’ indicates …
Figure 7 with 3 supplements
Cl- currents in the non-ciliated cells of the efferent ductules through CFTR.
(A, D and F) Corresponding I-V curves of the whole-cell Cl- IADGRG2-ED currents recorded in Figure 6 and (A, D and F) Corresponding I-V curves of the whole-cell Cl- IADGRG2-ED currents recorded in Fi…
Figure 7—figure supplement 1
Effects of different stimulators or inhibitors of osmotic drivers on I ADGRG2-ED Cl- currents of efferent ductule cells derived from Adgrg2-/Y mice and their wild type littermates.
(A) The whole cell Cl- current of I ADGRG2-ED elicited by voltage steps between −100 mV and +100 mV in a representative ADGRG2-promoter-RFP-labeled efferent ductule cells derived from the Adgrg2-/Y …
Figure 7—figure supplement 2
Effects of Cl- concentration change and CFTRinh-172 on the IADGRG2-ED Cl- currents.
(A) The whole cell Cl- current of IADGRG2-ED elicited by voltage steps between −100 mV and +100 mV in a representative ADGRG2-promoter-RFP-labeled efferent ductule cells derived from the Adgrg2-/Y …
Figure 7—figure supplement 3
Effects of CFTR knocked down on the I ADGRG2-ED Cl- currents.
(A) The whole cell Cl- current of I ADGRG2-ED elicited by voltage steps between −100 mV and +100 mV of primary efferent ductile cells after CFTR-siRNA or Scramble-siRNA treatment. (B) Corresponding …
Figure 8 with 3 supplements
Gq activity regulated Cl- current and pH homeostasis in the efferent ductules.
(A) Intracellular pH (pHi) of the ligated efferent ductules from WT (n = 9) mice or Gnaq+/- (n = 9) mice was measured by carboxy-SNARF. (B). The whole-cell Cl- current of the IADGRG2-ED elicited by …
Figure 8—figure supplement 1
Effects of G protein signaling on the I ADGRG2-ED Cl- currents.
(A) The whole cell Cl- current of IADGRG2-ED elicited by voltage steps between −100 mV and +100 mV in a representative ADGRG2-promoter-RFP-labeled efferent ductule cells derived from the Gnaq+/- …
Figure 8—figure supplement 2
Gq is localized in the ADGRG2 expressed cells, but not the acetylated-tubulin-labeled cells in efferent ductules.
(A) Bar graph representation and statistical analyses of co-localization of Gq and ADGRG2 in WT male mice efferent ductules (corresponding to Figure 8E), n = 3 mice per group; 4–10 random areas were …
Figure 8—figure supplement 3
The expression of ADGRG2, CFTR, Gs, Gq, β-arrestin-1, β-arrestin-2 in efferent ductules, brain and liver tissue of WT and Adgrg2-/Y mice.
(A) Western blot analysis of ADGRG2, CFTR, Gs, Gq, β-arrestin-1, β-arrestin-2 expression in efferent ductules, brain and liver tissue of WT and Adgrg2-/Y mice. A representative western blot from at …
Figure 9 with 3 supplements
β-arrestin-1 is required for fluid reabsorption in the efferent ductules via scaffolding ADGRG2/CFTR complex formation.
(A) Diameters of the luminal ductules derived from WT (n = 12), Adgrg2-/Y (n = 12) or Arrb1-/- (n = 15) mice. (B) Diameters of the luminal ductules derived from WT (n = 12), Adgrg2-/Y (n = 12) or Arr…
Figure 9—figure supplement 1
Western blot analysis of β-arrestin1/2 expression in the efferent duct tissue.
(A,C) Western blot analysis of β-arrestin1/2 expression in the efferent duct tissue of WT and Arrb2-/-(A) or Arrb1-/-(C) mice. A representative western blot from at least three independent …
Figure 9—figure supplement 2
β-arrestin-1 is an essential component in a signaling complex encompassing the ADGRG2 and CFTR in efferent ductules.
(A) Co-localization of Ezrin (red fluorescence) and CFTR (sc-8909, Santa Cruz, green fluorescence) in male efferent ductules of the WT mice and Adgrg2-/Y mice. Scale bars, 50 μm. Analysis of Ezrin …
Figure 9—figure supplement 3
The complex formation between ADGRG2, β-arrestin-1 and CFTR in HEK293 cells.
(A) HEK293 cells were transfected with equal amount plasmids encoding ADGRG2, CFTR, β-arrestin-1 or β-arrestin-2 plasmids. The Flag- ADGRG2 were pulled down by M2-Flag beads and the associated CFTR, …
Figure 10 with 5 supplements
ADGRG2 upregulates CFTR Cl- currents through G protein signaling.
(A) Whole-cell Cl- currents recorded with a CsCl pipette solution in HEK293 cells transfected with plasmids encoding ADGRG2 or/and CFTR, with or without CFTR inhibitor CFTRinh-172(10 μM) or its …
Figure 10—figure supplement 1
Co-localization analysis of ADGRG2 and CFTR in HEK293 cells.
(A) Co-localization of ADGRG2 (red fluorescence) and CFTR (green fluorescence) in HEK293 cells. Scale bars, 20 μm. A representative figure from at least three independent experiments was shown. (B) …
Figure 10—figure supplement 2
Construction and expression of ADGRG2-full length (ADGRG2FL) and a truncated form ADGRG2β.
(A–C) Construction and expression of ADGRG2-full length (ADGRG2FL) and a truncated form ADGRG2β. (A) Schematic illustration of the structure of the ADGRG2FL and the ADGRG2β used in the current …
Figure 10—figure supplement 3
Overexpression of ADGRG2FL and ADGRG2β lead to constitutive increased cellular cAMP levels.
(A–C) Overexpression of ADGRG2FL leads to constitutively increased intracellular cAMP levels. HEK293 cells were co-transfected with the GloSensor plasmid and the control pcDNA3.1 vector or the …
Figure 10—figure supplement 4
Overexpression of ADGRG2FL and ADGRG2β have constitutive Gq-NFAT signaling activities.
(A–B) Dose-dependent effect of ADGRG2FL (A) or ADGRG2β (B) overexpression on the luciferase activity of the NFAT-DLR. *p<0.05, **p<0.01, ***p<0.001, the ADGRG2 transfected cells were compared to …
Figure 10—figure supplement 5
ADGRG2 upregulates CFTR Cl- currents and Cl- efflux through G protein signaling.
(A) Corresponding bar graph of average reversal potential(Erev) (±s.e.m., n = 6 for each condition) in HEK293 cells transfected with plasmids encoding ADGRG2 or/and CFTR，with or without CFTR …
Figure 11 with 3 supplements
Key mutations of ADGRG2 downregulates CFTR Cl- currents through G protein signaling.
(A) Schematic representation of the location of the selected ADGRG2 mutants in intracellular loop 2 and loop 3 of ADGRG2. (B) Effects of the overexpression of ADGRG2 (n = 6) and its mutations (n = 6)…
Figure 11—figure supplement 1
Sequence alignment of the transmembrane domains of ADGRG2 (Homo sapiens, Mus musculus, Rattus norvegicus), β2AR (H. sapiens, M. musculus, and R. norvegicus), and GPR126 (H. sapiens).
The ADGRG2 mutation sites studied in the current work were highlighted. The green color indicated that the mutants caused both Gs and Gq defects of ADGRG2 coupling; the yellow color indicated that …
Figure 11—figure supplement 2
Western blot and ELISA analysis of the expression of these mutants in the cell membrane.
(A) Western blot of ADGRG2 WT and its mutations (HM696AA, H696A, M697A, Y698A, K703A, V704A, F705A and Y708A in intracellular loop 2; and QL798AA and RK803EE in intracellular loop 3). Representative …
Figure 11—figure supplement 3
Corresponding bar graph of average reversal potential(Erev) (±s.e.m., n = 6 for each condition) recorded in Figure 11D–11E and calculated Nernst potential.
ns., no significant difference; the Erev were compared with calculated Nernst potential.
Figure 12 with 1 supplement
Conditional expression of ADGRG2 wild-type or its selective G-subtype signaling mutants in the efferent ductules in Adgrg2-/Y mice and their effects on the morphology, sperm maturation of efferent ductules.
(A) Schematic representation of the mouse ADGRG2 promoters used in the rescue experiment. (B) Representative hematoxylin-eosin staining of the WT mice, Adgrg2-/Y mice or Adgrg2-/Y mice infected with …
Figure 12—figure supplement 1
Effect of the conditional expression of ADGRG2-WT or its selective G-subtype signaling mutants on the rescue of reproductive defects in Adgrg2-/Y mice.
(A) The enlarged images of the ADGRG2 expression in the epididymal initial segment at 3 weeks after the injection of the lentivirus of ADGRG2-WT or mutants. Scale bars, 100 μm. (B) Photographs of …
Figure 13
Effects of conditional expression of ADGRG2 wild-type or its selective G-subtype signaling mutants in Adgrg2-/Y mice on the fluid reabsorption of efferent ductules.
(A) Effects of the expression of the ADGRG2-WT adenovirus on the diameter of the ligated efferent ductules derived from the WT or Adgrg2-/Y mice. (B–F) Effects of the expression of adenovirus …
Figure 14
Schematic diagram depicting the GPCR signaling pathway in the regulation fluid reabsorption in the efferent ductules.
The ADGRG2 and CFTR localized at cell plasma membrane, whereas Gs and Gq localize at the inner surface of non-ciliated cells. Deficiency of ADGRG2 in Adgrg2-/Y mice, reducing the Gq protein level by …
Author response image 1
Expression and localization of Gs in different types of efferent ductule cells.
(A) The Gs was expressed in both GPR64-expressed cells and non-GPR64-expressed cells revealed by co-immunostaining. (B) The Gs expression in GPR64-promoter labeled cells and non GPR64-promoter …
Tables
Table 1
Average reversal potential calculated at different Cl- concentrations for Figure 6C.
Average reversal potential(Erev) (±s.e.m., n = 8 for each condition) in Figure 6C and calculated Nernst potential at different Cl- concentrations. The Nernst equation was: Erev=-RT/Z [Ln (Cl-)in/(Cl-…
| Group | Erev[Cl-]o148.5 mM(mV) | Erev[Cl-]o48.5 mM(mV) |
|---|---|---|
| Nernst | −4.6 | 25.3 |
| WT | −4.0 ± 0.51 | 20.1 ± 2.52 |
| Adgrg2-/Y | −4.1 ± 0.36 | 19.4 ± 2.47 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Chemical compound, drug | PTX | Enzo | Cat#:BML-G100 | 100 ng/ml |
| Chemical compound, drug | U0126 | Sigma | Cat#:U120 | 10 μM |
| Chemical compound, drug | Ro 31–8220 | Adooq | Cat#:A13514 | 500 nM |
| Chemical compound, drug | NF449 | Tocris | Cat#:1391 | 1 μM |
| Chemical compound, drug | PKI14-22 | Adooq | Cat#:A16031 | 300 nM |
| Chemical compound, drug | H89 | Beyotime | Cat#:S1643 | 500 nM |
| Chemical compound, drug | bumetanide | Aladdin | Cat#:B129942 | 10 μM |
| Chemical compound, drug | Ani9 | Sigma | Cat#:SML1813 | 150 nM |
| Chemical compound, drug | Niflumic acid (NFA) | Aladdin | Cat#:N129597 | 20 μM |
| Chemical compound, drug | DIDS | Sigma | Cat#:D3514 | 20 μM |
| Chemical compound, drug | GlyH-101 | Adooq | Cat#:A13723 | 10 μM |
| Chemical compound, drug | CFTRinh-172 | Adooq | Cat#:A12897 | 10 μM |
| Chemical compound, drug | EGTA | Aladdin | Cat#:E104434 | 5 mM |
| Chemical compound, drug | SKF96365 | Sigma | Cat#:S7809 | 10 μM |
| Chemical compound, drug | Ruthenium red | Sigma | Cat#:R2751 | 10 μM |
| Chemical compound, drug | Nicardipine | Sigma | Cat#:N7510 | 20 μM |
| Chemical compound, drug | LaCl3 | Sigma | Cat#:449830 | 100 μM |
| Chemical compound, drug | IBMX | Sigma | Cat#:I7018 | 100 μM |
| Chemical compound, drug | U73122 | Sigma | Cat#:U6756 | 10 μM |
| Chemical compound, drug | Forskolin | Beyotime | Cat#:S1612 | 10 μM |
| Chemical compound, drug | PD123319 | Adooq | Cat#:A13201 | 1 μM |
| Chemical compound, drug | Candesartan | Adooq | Cat#:A10175 | 1 μM |
| Chemical compound, drug | Amiloride | Aladdin | Cat#:A129545 | 1 mM |
| Chemical compound, drug | Acetazolamide | Medchem express | Cat#:HY-B0782 | 500 μM |
| Peptide, recombinant protein | ANGII | China Peptides | 100 nM | |
| Commercial assay or kit | Carboxy SNARF−1, acetoxymethyl ester | Invitrogen | Cat#:C-1272 | 5 μM |
| Commercial assay or kit | Lipofectamine TM2000 | Invitrogen | Cat#:11668–019 | |
| Commercial assay or kit | Collagenase I | sigma | Cat#:C0130 | |
| Commercial assay or kit | cAMP ELISA kit | R and D systems | Cat#:KGE012B | |
| Commercial assay or kit | IP1 ELISA assay | Shanghai Lanpai Biotechnology Co., Ltd | Cat#:lp034186 | |
| Commercial assay or kit | The dual-luciferase reporter assay system | Promega | Cat#:E1960 | |
| Antibody | ADGRG2 antibody(rabbit polyclonal) | Sigma | RRID:AB_1078923 | |
| Antibody | ADGRG2 antibody(rabbit polyclonal) | Sigma | RRID:AB_2722557 | |
| Antibody | ADGRG2 antibody(sheep polyclonal) | R and D systems | RRID:AB_2722556 | |
| Antibody | CFTR antibody(goat polyclonal) | Santa Cruz | RRID:AB_638427 | |
| Antibody | CFTR antibody(rabbit polyclonal) | Proteintech | RRID:AB_2722558 | |
| Antibody | Gq antibody(goat polyclonal) | Santa Cruz | RRID:AB_2279038 | |
| Antibody | Gq antibody(rabbit polyclonal) | Proteintech | RRID:AB_2111647 | |
| Antibody | Flag antibody(mouse monoclonal) | Sigma | RRID:AB_259529 | |
| Antibody | HA antibody(mouse monoclonal) | Santa Cruz | RRID:AB_627809 | |
| Antibody | GAPDH(rabbit monoclonal) | Cell Signaling | RRID:AB_10622025 | |
| Antibody | Gs antibody(rabbit polyclonal) | Proteintech | RRID:AB_2111668 | |
| Antibody | Gi antibody(mouse monoclonal) | Santa Cruz | RRID:AB_2722559 | |
| Antibody | β-arrestin-1 antibody(rabbit polyclonal) | Dr R.J. Lefkowitz | A1CT | |
| Antibody | β-arrestin-2 antibody(rabbit polyclonal) | Dr R.J. Lefkowitz | A2CT | |
| Antibody | ANO1 antibody(rabbit polyclonal) | Proteintech | RRID:AB_2722560 | |
| Antibody | Ezrin antibody(rabbit polyclonal) | Proteintech | RRID:AB_2722561 | |
| Antibody | Acetylated Tubulin(Lys40) Antibody(mouse monoclonal) | Proteintech | RRID:AB_2722562 | |
| Antibody | Donkey anti-sheep IgG(H + L) (secondary antibody) | Abcam | RRID:AB_2716768 | |
| Antibody | Donkey anti-rabbit IgG(H + L) (secondary antibody) | Invitrogen | RRID:AB_2534017 | |
| Antibody | Donkey anti-mouse IgG(H + L) (secondary antibody) | Invitrogen | RRID:AB_141607 | |
| Antibody | Donkey anti-goat IgG(H + L) (secondary antibody) | Invitrogen | RRID:AB_142672, RRID:AB_141788 | |
| Antibody | HRP-conjugated Affinipure Rabbit Anti-Sheep IgG(H + L) | Proteintech | RRID:AB_2722563 | |
| Antibody | HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) | Proteintech | RRID:AB_2722564 | |
| Antibody | HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) | Proteintech | RRID:AB_2722565 |
-
All other chemicals or reagents were from Sigma unless otherwise specified.
Additional files
-
Supplementary file 1
Primers for the Quantitative RT-PCR (qRT-PCR) analysis of mRNA transcription profiles of G protein subtypes and β-arrestins.
- https://doi.org/10.7554/eLife.33432.044
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Supplementary file 2
Primers for the Quantitative RT-PCR (qRT-PCR) analysis of mRNA transcription profiles of potential osmotic drivers including selective ion channels and transporters.
- https://doi.org/10.7554/eLife.33432.045
-
Supplementary file 3
Primers for the construction of ADGRG2FL mutants (HM696AA, H696A, M697A, Y698A, K703A, V704A, F705A, Y708A, QL798AA, RK803EE).
- https://doi.org/10.7554/eLife.33432.046
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Transparent reporting form
- https://doi.org/10.7554/eLife.33432.047
