Establishment of a chimeric reporting system for the universal detection and high-throughput screening of G protein-coupled receptors

Biosensors & Bioelectronics
Chun-Jen WangChing-Wei Luo

Abstract

G proteins, further divided into four subfamilies (G(s), G(q), G(12) and G(i)) based on their Galpha subunits, are the primary components activated by G protein-coupled receptors (GPCRs). Current GPCR assays are limited to the evaluation of selective Galpha signaling and do not allow comprehensive screening for orphan GPCRs without a known coupled Galpha. Therefore, our aim was to design a chimeric reporting system that covers responses from all Galpha subfamilies simultaneously. Because G(s) activates cAMP response element (CRE)-driven genes whereas G(q) and G(12) activate serum response element (SRE)-driven genes, we therefore incorporated 2x CRE and 5x SRE (2CRE5SRE) into a promoter for driving luciferase expression. To further report G(i) signals, a 2CRE5SRE-driven chimeric G(qi), in which the C-terminus of G(q) is replaced by that of G(i), was integrated to switch the responses of G(i)-coupled GPCRs to the G(q) signaling. The novel reporter system showed a strong signal amplification when activated by neuromedin U receptor 1 (mainly activates G(q)), neuromedin U receptor 2 (mainly activates G(i)) or luteinizing hormone receptor (mainly through the G(s) and G(q) pathways). In addition, 293T cells stably carrying our reporte...Continue Reading

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Citations

May 7, 2010·The AAPS Journal·Akihiko OzawaWesley K Kroeze
Dec 15, 2015·Frontiers in Cardiovascular Medicine·Danielle KamatoPeter J Little
Aug 6, 2015·Archives of Pharmacal Research·Yue ZhuMing-Wei Wang
Nov 2, 2012·American Journal of Physiology. Endocrinology and Metabolism·Wei-Yu LeeChing-Wei Luo

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