Figure 5. 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 CFTRinh-172. (B) qRT-PCR analysis of CFTR levels in the efferent ductules of WT (n = 3) or Adgrg2^-/Y (n = 3) mice. (C) Co-localization of ADGRG2 (red fluorescence) and CFTR (sc-8909, Santa Cruz, green fluorescence) in the male efferent ductules of WT mice. Scale bars, 50 μm. (D) Analysis of ADGRG2 and CFTR fluorescence intensities by Pearson’s correlation analysis. The Pearson's correlation coefficient was 0.76. (E) Immunofluorescence staining of ADGRG2 (red fluorescence) and CFTR (sc-8909, Santa Cruz, green fluorescence) in the efferent ductules of Adgrg2^-/Y mice. Scale bars, 50 μm. (F) Co-localization of ADGRG2 (red fluorescence) and ezrin (green fluorescence) in the male efferent ductules of WT mice. Scale bars, 50 μm. (G) Analysis of ADGRG2 and ezrin fluorescence intensities by Pearson’s correlation analysis. The Pearson's correlation coefficient was 0.69. (H) ADGRG2 was immunoprecipitated with an anti-ADGRG2 antibody from the male efferent ductules of WT mice or Adgrg2^-/Y mice, and co-precipitated CFTR, Gs, Gq, β-arrestin-1, β-arrestin-2 and Gi-1/2/3 levels were examined by using specific corresponding antibodies (CFTR antibody:20738–1-AP, Proteintech). (5A-5B) *p<0.05, **p<0.01, ***p<0.001, Adgrg2^-/Y mice compared with WT mice. #p<0.05, ##p<0.01, ###p<0.001. Treatment with selective inhibitors or stimulators was compared with control vehicles. n.s., no significant difference. At least three independent biological replicates were performed for Figure 5A–B.

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 6')-carboxy-10-dimethylamino-3-hydroxy-spiro[7H-benzo[c]xanthene-7,1'(3 hr)-isobenzofuran]−3'-one (carboxy SNARF-1)). (B–D) Intracellular pH (pHi) of the ligated efferent ductules with or without bicarbonate(25 mM)(B), Acetazolamide(500 μM)(C), Amiloride(1 mM)(D) from WT mice and Adgrg2^-/Y mice. (B–D) *p<0.05, selective inhibitors or stimulators treated were compared with control vehicles.

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, Santa Cruz, green fluorescence) and acetylated-tubulin (yellow fluorescence) in WT male mice efferent ductules. Scale bars, 50 μm. A representative image was selected from at least three independent experiments. (C) Bar graph representation and statistical analyses of co-localization cells of CFTR and acetylated-tubulin in WT male mice efferent ductules (related to (B)).

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.