A Novel Cyclic Adenosine Monophosphate–Responsive Luciferase Reporter Incorporating a Nonpalindromic Cyclic Adenosine Monophosphate Response Element Provides Optimal Performance for Use in G Protein–Coupled Receptor Drug Discovery Efforts

Abstract

The authors report the characterization of a novel cyclic adenosine monophosphate (cAMP)-responsive luciferase (Luc) reporter that exhibits optimal performance in high-throughput screens of agonist binding at G protein-coupled receptors (GPCRs). This reporter (RIP1-CRE-Luc) incorporates a nonpalindromic cAMP response element (CRE) originally identified within the 5′ promoter of the rat insulin 1 gene (RIP1). When multimerized and fused to the coding sequence of firefly luciferase, the CRE of RIP1 allows for the efficient activation of luciferase expression by cAMP-elevating agents or by cAMP itself. Of primary importance is the demonstration that RIP1-CRE-Luc does not exhibit the relatively high levels of basal luciferase activity inherent to reporters incorporating the palindromic CRE first identified in the somatostatin gene promoter. Furthermore, studies of HEK cells expressing class II GPCRs for the cAMP-elevating hormones GLP-1, GIP, and glucagon demonstrate that RIP1-CRE-Luc affords a much wider dynamic range of activation upon exposure to agonist. Such properties of RIP1-CRE-Luc indicate its usefulness as a new and powerful tool for the identification of small-molecule compounds with receptor-stimulating actions or for the identification of constitutively active orphan receptors with cAMP-signaling properties. (Journal of Biomolecular Screening 2007:740-746)

Stimulation of Proglucagon Gene Expression by Human GPR119 in Enteroendocrine L-cell Line GLUTag

“…The RIP1-CRE-Luc reporter generated in our laboratory (20) consists of 4 multimerized nonpalindromic cAMP response elements (CREs) found within the rat insulin I gene promoter (RIP1) fused to the coding sequence of firefly luciferase in pLuc-MCS. The A-CREB expression plasmid (21) …”

Development of an Assay for High-Throughput Energy Expenditure Monitoring in the Zebrafish

“…II; SignaGen Laboratories, Rockville, MD) with a cre-luciferase plasmid kindly provided by Dr. George Holz 14 and either the zmc3r or hmc3r (pcDNA3.1+ vector, Invitrogen, Carlsbad, CA) with 0.12 lg total plasmid in each well of a 96-well plate. The zmc3r plasmid has been previously employed for studies in our laboratory.…”

60 YEARS OF POMC: Melanocortin receptors: evolution of ligand selectivity for melanocortin peptides

“…4. All cells were transfected with the cre-luc cAMP reporter construct (Chepurny & Holz 2007). The transfected cells were stimulated with NDP-MSH (New England Peptide) at concentrations ranging from 10 -12 to 10 -6 M. Data are presented as mean ± s.e.m.…”

“…In addition, single alanine mutants of hMC2R were also made by GenScript, and the mutant cDNAs were individually co-transfected with mMRAP1 in CHO cells. The list of single alanine mutants is presented in panel A. CHO cells were transfected with the following plasmids: either human MC2R or human single alanine mutant MC2R, mouse mrap1, and the cAMP reporter plasmid cre-luc (Chepurny & Holz 2007) using a Cell Line Nucleofector Kit (Amaxa, Inc., Allendale, NJ, USA; www.lonza.com) as described by Liang et al (2011). After 48 h, the transfected cells were stimulated with hACTH(1-24) (New England Peptide, Gardner, MA, USA) in serum-free CHO media for 4 h at 37°C at concentrations ranging from 10 -7 to 10 -13 M. After a 4-h incubation period, 100 µL of Bright-Glo Luciferase Assay System (Promega) was applied to each well and incubated at room temperature for 5 min.…”