Review
Reporter-gene systems for the study of G-protein-coupled receptors

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Abstract

Reporter-gene assays offer an alternative to biochemical assays for following signal transduction pathways from receptors at the cell surface to nuclear gene transcription in living cells. Specific reporter-gene systems are now available for the study of ligand activity at Gαi/o, Gαs and Gαq G-protein-coupled receptors. In recent years reporter genes have been applied in academia and industry to the study of ligand efficacy and affinity in recombinant and primary cell lines using a variety of colour, fluorescent or luminescent read-outs.

Section snippets

Introduction — what is a reporter gene?

A reporter gene is a sequence of DNA whose product is synthesised in response to activation of the signalling cascade under investigation. The DNA sequence consists of a promoter, where transcription factors bind to control transcription, a reporter gene and a transcription stop signal. The choice of promoter element dictates the sensitivity and specificity of the reporter. The reporter must have low basal expression and a large transcriptional response following receptor activation, offer a

Signalling from the cell surface to the nucleus

GPCRs interact with heterotrimeric G proteins to regulate a range of second messenger pathways to enable communication from the cell surface to the nucleus. Upon activation of the receptor, the G protein dissociates into α and βγ subunits both of which stimulate intracellular signalling pathways [1]. GPCRs coupled to the Gαq family of G proteins stimulate the enzyme phospholipase Cβ (PLCβ). PLCβ cleaves membrane phospholipids to produce inositol-1,4,5-trisphosphate (IP3), which mobilises

Choice of promoter

The choice of promoter depends on the nature of the signalling pathway under study. Both natural and synthetic promoter elements have been used in reporter-gene assays. Natural promoter elements, such as the c-fos promoter, contain binding sites for a number of transcription factors (Fig. 2) and therefore the regulation of such promoters tends to be under the control of a complex range of signal transduction events [2]. To generate reporter assays specific for single signalling cascades

Applications and limitations of reporter genes

Reporter-gene assays are used to accurately determine the affinities of antagonists and the efficacy and relative potencies of agonists at GPCRs and have been used to study partial agonist activity, constitutive activity and inverse agonism (Table 1). Reporter-gene assays involve significant signal amplification between ligand binding and transcription. This is useful in that it allows the characterisation of partial agonists, for example at the 5-HT1B serotonin receptor and the D2 dopamine

Conclusions

Because of their relative simplicity reporter-gene assays are in widespread use for the study of ligand efficacy at GPCRs. Specific promoters have been developed for different classes of receptor and a variety of reporters are available that allow the detection of agonist activity in lysed or live cells. The development of high-throughput confocal imaging equipment [48•] and of reporters such as β-lactamase that are amenable to noninvasive assay in living cells, will enable the temporal

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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