Dataset Information

Adenosine Receptor A1-A2a Heteromers Regulate EAAT2 Expression and Glutamate Uptake via YY1-Induced Repression of PPAR? Transcription.

ABSTRACT: Adenosine receptors A1 (A1AR) and A2a (A2aAR) play an important role in regulating glutamate uptake to avoid glutamate accumulation that causes excitotoxicity in the brain; however, the precise mechanism of the effects of A1AR and A2aAR is unclear. Herein, we report that expression of the A1AR protein in the astrocyte membrane and the level of intracellular glutamate were decreased, while expression of the A2aR protein was elevated in cells exposed to oxygen-glucose deprivation (OGD) conditions. Coimmunoprecipitation (Co-IP) experiments showed that A1AR interacts with A2aAR under OGD conditions. The activation of A1AR and inactivation of A2aAR by 2-chloro-N6-cyclopentyladenosine (CCPA) and SCH58251, respectively, partly reversed OGD-mediated glutamate uptake dysfunction, elevated EAAT2, and PPAR? protein levels, and suppressed the expression of Ying Yang 1 (YY1). Both the silencing of YY1 and the activation of PPAR? upregulated EAAT2 expression. Moreover, YY1 silencing elevated the PPAR? level under both normal and OGD conditions. Histone deacetylase (HDAC)1 was found to interact with YY1, and HDAC1 silencing improved PPAR? promoter activity. Taken together, our findings suggest that A1AR-A2aAR heteromers regulate EAAT2 expression and glutamate uptake through the YY1-mediated recruitment of HDAC1 to the PPAR? promoter region.

SUBMITTER: Hou X

PROVIDER: S-EPMC7079221 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Adenosine Receptor A1-A2a Heteromers Regulate EAAT2 Expression and Glutamate Uptake via YY1-Induced Repression of PPAR<i>γ</i> Transcription.

Hou Xianhua X Li Yuan Y Huang Yuanyuan Y Zhao Huan H Gui Li L

PPAR research 20200306

Adenosine receptors A1 (A1AR) and A2a (A2aAR) play an important role in regulating glutamate uptake to avoid glutamate accumulation that causes excitotoxicity in the brain; however, the precise mechanism of the effects of A1AR and A2aAR is unclear. Herein, we report that expression of the A1AR protein in the astrocyte membrane and the level of intracellular glutamate were decreased, while expression of the A2aR protein was elevated in cells exposed to oxygen-glucose deprivation (OGD) conditions. ...[more]

PMID: 32206061

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Project description:Conventional methods to measure ligand-receptor binding parameters typically require radiolabeled ligands as probes. Despite the robustness of radioligand binding assays, they carry inherent disadvantages in terms of safety precautions, expensive synthesis, special lab requirements, and waste disposal. Mass spectrometry (MS) is a method that can selectively detect ligands without the need of a label. The sensitivity of MS equipment increases progressively, and currently, it is possible to detect low ligand quantities that are usually found in ligand binding assays. We developed a label-free MS ligand binding (MS binding) assay on the adenosine A(1) and A(2A) receptors (A(1)AR and A(2A)AR), which are well-characterized members of the class A G protein-coupled receptor (GPCR) family. Radioligand binding assays for both receptors are well established, and ample data is available to compare and evaluate the performance of an MS binding assay. 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX) and 4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol (ZM-241,385) are high-affinity ligands selective for the A(1)AR and A(2A)AR, respectively. To proof the feasibility of MS binding on the A(1)AR and A(2A)AR, we first developed an MS detection method for unlabeled DPCPX and ZM-241,385. To serve as internal standards, both compounds were also deuterium-labeled. Subsequently, we investigated whether the two unlabeled compounds could substitute for their radiolabeled counterparts as marker ligands in binding experiments, including saturation, displacement, dissociation, and competition association assays. Furthermore, we investigated the accuracy of these assays if the use of internal standards was excluded. The results demonstrate the feasibility of the MS binding assay, even in the absence of a deuterium-labeled internal standard, and provide great promise for the further development of label-free assays based on MS for other GPCRs.

Dataset Information

Adenosine Receptor A1-A2a Heteromers Regulate EAAT2 Expression and Glutamate Uptake via YY1-Induced Repression of PPAR? Transcription.

Publications

Adenosine Receptor A1-A2a Heteromers Regulate EAAT2 Expression and Glutamate Uptake via YY1-Induced Repression of PPAR<i>γ</i> Transcription.

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