Generation of a bioactive neuropeptide in a cell-free system

Jiang, Chunmiao; Chen, Guifen; Zeng, Xuemei; Ouyang, Keqing; Hu, Yinghe

doi:10.1016/s0003-2697(03)00040-x

Cited by 8 publications

(8 citation statements)

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“…To identify the endogenous ligand(s) for different GPCRs, a variety of peptides and peptide‐enriched tissue extracts were tested in a luciferase‐reporter assay (Jiang et al , 2003). For this purpose CV‐1 cells were co‐transfected with plasmids bearing the GPCR of interest and the luciferase reporter gene.…”

Section: Resultsmentioning

confidence: 99%

“…As control served an empty vector or another GPCRs. Stimulation of GPCRs with a cognate ligand should result in an increase (Gs, Gq) or a decrease (Gi) of the forskolin‐induced luminescence signals (Jiang et al , 2003). The transfection efficiency of CV‐1 cells, transiently expressing GPR75 was in the range of 25–30% as assessed by GPR75‐EGFP fluorescence.…”

Section: Resultsmentioning

confidence: 99%

“…After 24 h, the cells were transiently transfected with human GPR75 or vector cDNA (as control) and the reporter‐gene construct using lipofectamine 2000 (Invitrogen). The reporter contains three multiple‐response elements (MREs), a cAMP‐response element (CRE) and a serum‐response element (SRE) upstream of the luciferase gene (Jiang et al , 2003). The ratio of the receptor or vector to the reporter‐gene construct was 4:1.…”

Section: Methodsmentioning

confidence: 99%

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RANTES stimulates Ca²⁺ mobilization and inositol trisphosphate (IP₃) formation in cells transfected with G protein‐coupled receptor 75

Ignatov

Robert

Gregory‐Evans

et al. 2006

British J Pharmacology

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Background and purpose: RANTES is an inflammatory chemokine with a critical role in T-lymphocyte activation and proliferation. Its effects are mediated through G protein-coupled heptahelical receptors (GPCRs). We show for the first time that RANTES activates the orphan G protein-coupled receptor 75 (GPR75). Experimental approach: To identify a ligand for GPR75 we have used three different and independent methods, namely luciferase assay, bioluminescence assay and IP 3 accumulation assay. Key results: Treatment of cells expressing GPR75 with subnanomolar concentrations of RANTES led to stimulation of the luciferase activity in a reporter-gene assay, an increase in inositol trisphosphate, and intracellular Ca 2 þ . The latter effect was blocked by the phospholipase-C inhibitor (PLC) U73122 indicating that Gq proteins mediate GPR75 signaling. RANTES enhanced the phosphorylation of AKT and mitogen-activated protein kinase (MAPK) in GPR75-transfected cells and this effect was blocked by the PLC inhibitor U73122 and the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. The hippocampal cell line HT22, which expresses GPR75 endogenously, but not the other known RANTES receptors, was used to study the effects of RANTES and GPR75 on neuronal survival. Treatment of HT22 cells with RANTES significantly reduced the neurotoxicity of amyloid-b peptides, by activating PLC and PI3K. Conclusions and implications: This demonstrate clearly and undoubtedly the ability of RANTES to act on GPR75. Defects in the RANTES/GPR75-signaling pathway may contribute to neuroinflammatory and neurodegenerative processes as observed in Alzheimer's disease.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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RANTES stimulates Ca²⁺ mobilization and inositol trisphosphate (IP₃) formation in cells transfected with G protein‐coupled receptor 75

Ignatov

Robert

Gregory‐Evans

et al. 2006

British J Pharmacology

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“…Bovine rhodopsin [4], bovine opsin [5] and squid rhodopsin [6,7], whose crystal structures have been solved, are expressed in large amounts endogenously, and therefore can be obtained from natural sources, whereas other GPCRs cannot be purified in large amounts from natural tissues because of their low endogenous expression levels. In addition to insect cells [8-16], various expression systems can be applied to obtain a high yield of GPCRs, such as Escherichia coli [17-21], Saccharomyces cerevisiae [22,23], Pichia pastoris [24-30], mammalian cell lines [31-33], and a cell-free translation system [34-39]. We previously identified 25 GPCRs expressed by P. pastoris [29], which led us to suggest that P. pastoris is a suitable host for GPCR crystal structural studies.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

et al. 2011

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BackgroundVarious protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.ResultsThe ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells.ConclusionCompared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs.

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“…Recently, we developed a universal functional assay for GPCR [23] . In the present report, we further modified this functional assay for human D1-like agonist screening.…”

Section: Introductionmentioning

confidence: 99%

Identification of human dopamine D1-like receptor agonist using a cell-based functional assay1

Jiang

Ouyang

Cai

et al. 2005

Acta Pharmacologica Sinica

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Aim: To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library. Methods: Synthetic responsive elements 6×cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings. Results: A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors. The EC 50 values of SBG492 were 342.7 µg/mL for the D1 receptor and 31.7 µg/mL for the D5 receptor. Conclusion: We have established a cell-based assay for high-throughput drug screening to identify D1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered. These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.

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Generation of a bioactive neuropeptide in a cell-free system

Cited by 8 publications

References 38 publications

RANTES stimulates Ca²⁺ mobilization and inositol trisphosphate (IP₃) formation in cells transfected with G protein‐coupled receptor 75

RANTES stimulates Ca²⁺ mobilization and inositol trisphosphate (IP₃) formation in cells transfected with G protein‐coupled receptor 75

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

Identification of human dopamine D1-like receptor agonist using a cell-based functional assay1

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Generation of a bioactive neuropeptide in a cell-free system

Cited by 8 publications

References 38 publications

RANTES stimulates Ca2+ mobilization and inositol trisphosphate (IP3) formation in cells transfected with G protein‐coupled receptor 75

RANTES stimulates Ca2+ mobilization and inositol trisphosphate (IP3) formation in cells transfected with G protein‐coupled receptor 75

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

Identification of human dopamine D1-like receptor agonist using a cell-based functional assay1

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RANTES stimulates Ca²⁺ mobilization and inositol trisphosphate (IP₃) formation in cells transfected with G protein‐coupled receptor 75

RANTES stimulates Ca²⁺ mobilization and inositol trisphosphate (IP₃) formation in cells transfected with G protein‐coupled receptor 75