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First published online April 23, 2007
doi: 10.1242/10.1242/jcs.001230

Journal of Cell Science 120, 1521-1528 (2007)
Published by The Company of Biologists 2007
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YWK-II protein as a novel Go-coupled receptor for Müllerian inhibiting substance in cell survival

Xueqian Yin1,*, Songying Ouyang1,*, Wenming Xu2,*, Xiaopeng Zhang1, Kin Lam Fok2, Hau Yan Wong2, Jiaping Zhang2, Xiaobo Qiu1, Shiying Miao1, Hsiao Chang Chan2,{ddagger} and Linfang Wang1,{ddagger}

1 National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China
2 Epithelial Cell Biology Research Center, Li Ka Shing Institute of Health Sciences, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China


Figure 1
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  		Fig. 1. Localization of the EGFP-YWK-II protein on the cell membrane visualized by laser confocal scanning microscopy. (a-c) CHO cells transiently transfected with pEGFP-N1 were fixed, permeabilized and stained with propidium iodide (PI). (a) EGFP-overexpressing CHO cells (green). (b) CHO cells stained with PI to delineate the nucleus (red). (c) superimposed image of a and b. (d-f) CHO cells transiently transfected with pEGFP-N1-YWK-II were fixed, permeabilized and stained with PI. (d) EGFP-YWK-II-overexpressing CHO cells (green). (e) CHO cells stained with PI to delineate the nucleus (red). (f) Superimposed image of d and e. (g-i) CHO cells were transiently transfected with pEGFP-N1-YWK-II, fixed and not permeabilized. (g) EGFP-YWK-II-overexpressing CHO cells (green). (h) EGFP-YWK-II protein overexpressed on the cell membrane with rabbit YWK-II antiserum as the primary antibody and TRITC-conjugated goat anti-mouse IgG as the second antibody (red). (i) Superimposed image of g and h. (j-l) CHO cells were transiently transfected with pEGFP-N1-YWK-II, fixed and not permeabilized. (j) EGFP-YWK-II-overexpressing CHO cells (green). (k) EGFP-YWK-II-overexpressing cells using rabbit preimmune serum as the primary antibody and TRITC-conjugated goat anti-mouse IgG as the secondary antibody showing negative staining. (l) Superimposed image of j and k.

 

Figure 2
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  		Fig. 2. Confirmation of YWK-II protein expression in the CHO cell line by western blotting. Electrophoresis pattern of lysates of CHO cell, CHO cells stably transfected with pEGFP-N1-YWK-II (pEGFP-YWK-II) or pEGFP-N1 (pEGFP), stained with rabbit YWK-II antibody. The total protein loaded on the gel was 20 µg. Only the 67 kDa protein in lysate obtained from stable CHO cells transfected with pEGFP-N1-YWK-II was detected by YWK-II antibody (molecular mass of recombinant EGFP-YWK-II protein, ~67 kDa).

 

Figure 3
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  		Fig. 3. Demonstration of involvement of YWK-II protein in MIS-induced ERK1/2 phosphorylation coupled to Go, Gobeta{gamma}, Ras and PKC. Cells were lysed and assayed for p-ERK1/2 and total ERK1/2 by western blotting. The averaged ratio values of triplicate experiments are presented with corresponding western blot results. Unless stated otherwise, the cells were starved for 16 hours prior to stimulation with 0.35 nM rMIS at 37°C for 5 minutes. Statistical analysis was performed using Student's t-test (two-tailed comparison of means for paired samples). (A) Enhanced rMIS-induced ERK1/2 phosphorylation in YWK-II-overexpressing CHO cells (pEGFP-N1-YWK-II) as compared to that in EGFP-overexpressing CHO cells (pEGFP-N1) and non-transfected CHO cells. Different concentrations of rMIS used: 0, 0.035 and 0.35 nM (P<0.01). (B) Concentration-dependent effect of rMIS on ERK1/2 phosphorylation in EGFP-YWK-II-overexpressing CHO cells. Different concentrations of rMIS used: 0, 0.0175, 0.035, 0.35, 3.5 and 35 nM. (C) Effect of PTX on rMIS-induced ERK1/2 phosphorylation in EGFP-YWK-II-overexpressing CHO cells. Cells were starved for 16 hours and pretreated with PTX (1 µg/ml) or left untreated for 24 hours prior to stimulation with rMIS (P<0.01). (D) Effects of C-terminal regions of Gi1/2, G{alpha}o1 and G{alpha}o2 on rMIS-induced ERK1/2 phosphorylation. pcDNA3.1(+)-G{alpha}i1/2, pcDNA3.1(+)-G{alpha}o1 and pcDNA3.1(+)-G{alpha}o2 were transfected into EGFP-YWK-II-overexpressing CHO cells for 24 hours (P<0.05). (E) Effect of betaARK1 on rMIS-induced ERK1/2 phosphorylation. The EGFP-YWK-II-overexpressing CHO cells were transfected with pCMV-betaARK1-C for 24 hours (P<0.05). (F) Effects of pCMV-RasN17 and pCMV-RasV12 on rMIS-induced ERK1/2 phosphorylation. EGFP-YWK-II-overexpressing CHO cells were transfected with pCMV-RasN17 and pCMV-RasV12 for 24 hours (P<0.05). (G) Effect of GF109203X on rMIS-induced ERK1/2 phosphorylation in EGFP-YWK-II-overexpressing CHO cells. Cells were starved for 16 hours and pretreated with GF109203X (3.5 µM) or left untreated for 2 hours prior to stimulation with rMIS (P<0.01). (H) Effect of pCMV-RasN17 and GF109203X on rMIS-induced ERK1/2 phosphorylation. EGFP-YWK-II-overexpressing CHO cells were transfected with pCMV-RasN17 for 24 hours. The cells were starved for 16 hours and treated with or without GF109203X (3.5 µM) for 2 hours prior to stimulation with rMIS (P<0.01).

 

Figure 4
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  		Fig. 4. Effect of rMIS on cell viability and apoptotic activity in EGPF-YWK-II-overexpressing CHO cells. (A) Effect of rMIS and YWK-II proteins on cell viability. Cells transfected with EPGFP-YWK-II protein or EGFP and non-treated CHO cells were treated with different concentrations of rMIS for 3 days and assayed using the MTT method. The values obtained with the control untreated groups were set at 100% (*P<0.01). (B) Effect of rMIS on p53 levels in CHO cells. The established EGFP-YWK-II-overexpressing (YWK-II) and EGFP-transfected (EGFP) CHO cells and non-transfected CHO controls were cultured to confluence and incubated in serum-free medium for 24 hours prior to stimulation with 0.35 nM rMIS for 5 minutes. (C) Effect of rMIS on caspase-3 levels in YWK-II-overexpressing CHO cells. The cells were treated with 0.35 nM rMIS in the presence of YWK-II antibody (YWK-II Ab, 150 ng) or control IgG (150 ng) followed by western blot analysis of pro-caspase-3, with beta-actin as the loading control.

 

Figure 5
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  		Fig. 5. Effect of rMIS and YWK-II antibody on mouse sperm viability and ERK activation. (A) Sperm viability in response to treatment with rMIS and YWK-II antibody. Sperm were preincubated with control IgG or YWK-II antibody (Ab) for 15 minutes prior to addition of equal volume of PBS or rMIS (0.075 µM) followed by further incubation for 10 minutes or 30 minutes. Sperm were mixed with 0.04% Trypan Blue for evaluation of viability. The data are the mean percentage of viable sperm from three experiments (*P<0.05). (B) Effect of YWK-II antibody on rMIS-induced ERK1/2 activation in mouse sperm. Sperm were preincubated with control IgG or YWK-II antibody for 15 minutes prior to addition of rMIS (0.075 µM) followed by further incubation for 30 minutes. The proteins were extracted for western blot analysis with total ERK1/2 as a loading control.

 

Figure 6
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  		Fig. 6. In vivo effect of YWK-II antibody on sperm count and apoptotic activity of mouse testis. (A) Adult BALB/c mice were injected with YWK-II antibody (Ab; 40 µg/ml) or control IgG as described in the Materials an Methods and cauda sperm were recovered 48 hours later and sperm count was analyzed by computer-assisted sperm analysis. The data are the mean number of sperm per testis from nine mice (*P<0.05, n=9). (B) In vivo effect of YWK-II antibody on expression level of nuclear p53 and cleaved caspase-3 in mouse testis, demonstrated by western blot analysis.

 

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© The Company of Biologists Ltd 2007