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Development of a Novel Assay Based on DnaE Intein for Quantitative Analysis of GPCRs Internalization

Author: ZhangYaPing
Tutor: ZhouNaiMing
School: Zhejiang University
Course: Biochemistry and Molecular Biology
Keywords: G-protein-coupled receptors quantitative analysis of intemalization functional assay DnaE intein Luciferase
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 24
Quote: 0
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Abstract


G-protein-coupled receptors（GPCRs） are involved in a wide range of physiological processes and diseases,and represents significant and attractive targets for drug discovery.GPCR internalization provides a G-protein-subtype-independent method to determine agonist-stimulated activation of receptors.Firstly a new split DnaE intein gene was cloned from the Cyanobacteria and characterized.Then we have developed a novel assay for quantitative analysis of GPCR internalization based on reconstitution of split fragments of Renilla Luciferase（RLuc） by protein trans-splicing with Npu-DnaE intein and the interaction between agonist-activated GPCR andβ-arrestins2.This assay system has been further validated with four functionally divergent GPCRs:the insect adipokinetic hormone receptor（AKHR）,the human nicotinic acid receptor（HM74a）,the human cannabinoid receptor 2（CB2） and the human Histamine 1 receptor（H1）.The EC50 data obtained for known agonists and antagonist are in close agreement with the results of previous reports,indicating that this assay system has sufficient sensitivity for quantization of GPCR internalization. This rapid and quantitative assay could therefore be used as a universal and functional cell-base assay for GPCR high-throughput screening for drug discovery.

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