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. 2023 Apr 17;10(16):2205993. doi: 10.1002/advs.202205993

Figure 4.

Figure 4

Adgrv1 Y6236fsX1 mutation attenuates the inhibitory effects on local cAMP/PKA‐regulated WHRN phosphorylation. a,b) Effects of WT ADGRV1 or Y6236fsX1 mutant on the forskolin (FSK)‐induced cAMP accumulation in HEK293 cells in the absence or presence of WHRN and USH2A. Both the time course of a) cAMP production and b) quantitative analysis were shown. Data were normalized to the maximal cAMP response in control cells transfected with empty vector pcDNA3.1 and are from three independent experiments (n = 3). c) Left: Time course of cAMP production indicated by average G/R emission in HEK293 cells expressing WHRN‐FluoSTEP‐ICUE probe, USH2A and ADGRV1 (WT or Y6326fsX1 mutant) following stimulation with 50 µM Fsk and 100 µM IBMX. Right: Summary of emission ratio change (△R/△Rmax ) for WHRN‐FluoSTEP‐ICUE (△R/△R max; see Experimental Section). Data were obtained from 11–15 cells from three independent experiments. The domain structure of the WHRN‐FluoSTEP‐ICUE probe was shown in Figure S4g, Supporting Information. d) Representative blotting showing the FSK‐stimulated phosphorylation levels of USH2A or WHRN in HEK293 cells transfected with Myc‐WHRN, Myc‐USH2A, and Flag‐ADGRV1 (WT or Y6236fsX1 mutant). HEK293 cells were cotransfected with different combinations of ALC components as indicated in the Figure. The cells were treated with 10 µM FSK or a control vehicle for 30 min before the lysates were immunoprecipitated by Myc antibody‐conjugated agarose. The phosphorylation levels of Myc‐tagged WHRN or USH2A were detected by phospho‐(Ser/Thr) PKA substrate antibody. Only the phosphorylation of WHRN but not USH2A was detected as indicated by the pWHRN bands. e) Quantitative analysis of FSK‐stimulated phosphorylation of WHRN in HEK293 cells. The data are correlated with Figure 4d and the band intensity of phosphorylated WHRN co‐immunoprecipitated with Myc antibody‐conjugated agarose in the HEK293 cells transfected with Myc‐WHRN/Myc‐USH2A and treated with 10 µM forskolin was used as the reference (normalized to 1). Data are from three independent experiments (n = 3). f) Representative blotting and g) quantitative analysis of WHRN phosphorylation levels in the lysates of cochlea isolated from WT or Y6236fsX1 mutant mice. The lysates were immunoprecipitated by WHRN antibody‐conjugated agarose and the phosphorylation levels were detected by phospho‐(Ser/Thr) PKA substrate antibody. Data were normalized to the WHRN phosphorylation level in the lysates of cochlea isolated from WT mice and are from three independent experiments (n = 3). h) The PKA phosphorylation sites on WHRN were identified by mass spectrometry analysis. HEK293 cells transfected with Flag‐WHRN were stimulated with 10 µM FSK or control vehicle for 30 min before the WHRN was immunoprecipitated by Flag antibody‐conjugated agarose. The bound proteins were eluted with buffer containing Flag peptide and subjected to mass spectrometry analysis. Data are correlated with Figure S5, Supporting Information. i) Mass spectrum of the WHRN peptide containing phosphorylated serine at S156. j) Representative blotting and k) quantitative analysis of PKA phosphorylation levels of WHRN in HEK293 cells transfected with Flag‐tagged WT WHRN or mutants. Data are normalized to the phosphorylation level of WT WHRN and are from three independent experiments (n = 3). l) Effects of different phospho‐mimic mutations or m) phospho‐deficient mutations of WHRN on the stability of USH2A. Data are correlated with Figure S4k,l, Supporting Information and are from three independent experiments (n = 3). n) Quantitative analysis of the Myc‐USH2A coimmunoprecipitated with Flag‐tagged WHRN (WT or phospho‐mimic mutants) in HEK293 cells. Data are correlated with Figure S4m, Supporting Information and are from three independent experiments (n = 3). Data information: b) *p < 0.05; HEK293 cells transfected with WT ADGRV1 compared with the control cells. ## p < 0.01; HEK293 cells transfected with ADGRV1/WHRN/USH2A or with Y6236fsX1 compared with those transfected with WT ADGRV1. ns: no significant difference; HEK293 cells transfected with Y6236fsX1/WHRN/USH2A compared with those transfected with Y6236fsX1 only. c) ***p < 0.001; HEK293 cells transfected with WHRN‐FluoSTEP‐ICUE probe, USH2A, and ADGRV1 (WT or mutant) compared with those transfected with WHRN‐FluoSTEP‐ICUE probe and USH2A. ### p < 0.001; HEK293 cells transfected with Y6236fsX1 compared with those transfected with WT ADGRV1. e) ***p < 0.001; HEK293 cells transfected with ADGRV1/WHRN/USH2A compared with those transfected with WHRN/USH2A. ### p < 0.001; HEK293 cells transfected with Y6236fsX1/WHRN/USH2A compared with those transfected with ADGRV1/WHRN/USH2A. g) ***p < 0.001; Y6236fsX1 mutant mice compared with WT mice. k–n) *p < 0.05; **p < 0.01; ***p < 0.001; ns: no significant difference; HEK293 cells transfected with WHRN mutants compared with those transfected with WT WHRN. The bars indicate the mean ± SEM values. Data were statistically analyzed using b,c,e,g,k,n) one‐way or l,m) two‐way ANOVA with Dunnett's post‐hoc test.