eLife
·
2018
·
IF 7.616
Dao-Lai Zhang,Yu-Jing Sun,Ming-Liang Ma,Yi-jing Wang,Hui Lin,Rui-Rui Li,Zong-Lai Liang,Yuan Gao,Zhao Yang,Dong-Fang He,Amy Lin,Hui Mo,Yu-Jing Lu,Meng-Jing Li,Wei Kong,Ka Young Chung,Fan Yi,Jian-Yuan Li,Ying-Ying Qin,Jingxin Li,Alex R B Thomsen,Alem W Kahsai,Zi-Jiang Chen,Zhi-Gang Xu,Mingyao Liu,Dali Li,Xiao Yu,Jin-Peng Sun
Luminal fluid reabsorption plays a fundamental role in male fertility. We demonstrated that the ubiquitous GPCR signaling proteins Gq and β-arrestin-1 are essential for fluid reabsorption because they mediate coupling between an orphan receptor ADGRG2 (GPR64) and the ion channel CFTR. A reduction in protein level or deficiency of ADGRG2, Gq or β-arrestin-1 in a mouse model led to an imbalance in pH homeostasis in the efferent ductules due to decreased constitutive CFTR currents. Efferent ductule dysfunction was rescued by the specific activation of another GPCR, AGTR2. Further mechanistic analysis revealed that β-arrestin-1 acts as a scaffold for ADGRG2/CFTR complex formation in apical membranes, whereas specific residues of ADGRG2 confer coupling specificity for different G protein subtypes, this specificity is critical for male fertility. Therefore, manipulation of the signaling components of the ADGRG2-Gq/β-arrestin-1/CFTR complex by small molecules may be an effective therapeutic strategy for male infertility.
研究领域:
分子生物学
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生殖生物学
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药理学
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产品关联
DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2021
·
IF 4.821
Xiaojie Song,Man-Man Lv,Qi-Yan Lv,Hui-Fang Cui,Jing Fu,Yu-Yang Huo
A novel and sensitive Helicobacter pylori assay strategy based on linear isothermal amplification reaction (LIAR) and DNAzymes catalyzed DNA sensor was developed. A 17 nt target DNA (tDNA) specific in the H. pylori genome was selected and used as primer in the LIAR. A template DNA was designed to produce an amplicon with Mg 2+ -dependent and RNA-cleaving DNAzyme (Md-Dz) activity. The amplicon (i.e. Md-Dz) was then detected with the DNA sensor. The DNA sensor was fabricated simply through immobilization of a hairpin DNA that contains both Md-Dz substrate and G-quadruplex DNAzyme (Gq-Dz), on gold electrodes. The Gq-Dz possesses peroxidase activity, catalyzing the electro-redox of the electrochemical signal probe hydroquinon. The LIAR, Md-Dz catalytic reaction, and DNA sensor fabrication and detection processes were characterized and optimized. The LIAR amplification efficiency is as high as ~5 × 10 5 fold, and the assay strategy is very sensitive, selective and repetitive. The differential pulse voltammetry (DPV) detection signals linearly correlate to the logarithmic tDNA concentration in the range of 0.1–10,000 fM, with a limit of detection (LOD) of 34 aM. The detection linear range for H. pylori genomic DNA is 2.1–67.2 pg, with an LOD of 1.3 pg . Muted DNA sequences and other pathogenic bacteria did not result in interferences. In addition, the DNA sensor is very stable in storage. More importantly, the DNA sensor and the assay strategy could be universally applicable to detect any biological sample and short DNA sequence of interests.
研究领域:
生物化学
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分析化学
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分子生物学
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生物技术
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DOI
高影响因子
Analytical Methods
·
2019
·Vol. 11
·期 42
·5398-5404
·
IF 2.378
Shengting Zhang,Mingrui Li,Dongbo Liu,Kunhao Qin,Yingying Xiang,Xiuling Ji,Congwen Li,Yunlin Wei
The human purinergic P2Y1 receptor (P2Y1R) belongs to the family of G-protein coupled receptors (GPCRs). Two stable cell lines, hP2Y1R-tGFP/U2OS and hP2Y1R-β-AR2-tGFP/U2OS, were generated with steady expression of fluorescent tGFP tagged P2Y1R or tGFP tagged β-arrestin2. The High Content Screening (HCS) technique was used to investigate P2Y1R mediated signal transduction processes based on the distribution of fluorescent receptors or the β-arrestin2/receptor complex. All procedures including aggregation, endolysis and resensitization that were involved in the interaction between the P2Y1 receptor and its ligand can be visualized using the tGFP labeled receptor combined with HCS technology in this study. ADP activated P2Y1R to induce a distinguished pattern of fluorescent vesicle formation inside both cell types. These two cell lines produced different peak fluorescence signals when activated by ADP at 100 μM, and the time to form the maximal vesicles was detected as 15 and 20 min respectively. The EC50 values of the concentration–response curves of ADP induced formation of fluorescent vesicles were 35.7 nM and 71.8 nM respectively. The ADP effects could be blocked by a P2Y1R specific antagonist MRS2179. The P2Y1R are coupled to Gq protein to activate PLC first, PLC cleave PIP2 into IP3 and DAG. And then DAG and Ca2+ activate PKC pathway, and finally the phospholipase D initiates β-arrestin2 mediated signal pathway. It took 30 and 45 min to complete P2Y1R re-sensitization in these two constructs respectively. Our results demonstrated that P2Y1R triggered downstream responses not only via heterotrimeric Gq proteins but also through β-arrestin2.
研究领域:
细胞生物学
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药理学
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分子生物学
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信号转导
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产品关联
DOI
高影响因子
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