Article

Innovative functional cAMP assay for studying G protein-coupled receptors: Application to the pharmacological characterization of GPR17

Medicinal Chemistry Unit, School of Pharmacy, University of Camerino, 62032, Camerino, Italy.
Purinergic Signalling (Impact Factor: 3.89). 07/2011; 7(4):463-8. DOI: 10.1007/s11302-011-9245-8
Source: PubMed

ABSTRACT

In this work, an innovative and non-radioactive functional cAMP assay was validated at the GPR17 receptor. This assay provides a simple and powerful new system to monitor G protein-coupled receptor activity through change in the intracellular cAMP concentration by using a mutant form of Photinus pyralis luciferase into which a cAMP-binding protein moiety has been inserted. Results, expressed as EC(50) or IC(50) values for agonists and antagonists, respectively, showed a strong correlation with those obtained with [(35)S]GTPγS binding assay, thus confirming the validity of this approach in the study of new ligands for GPR17. Moreover, this method allowed confirming that GPR17 is coupled with a G(αi).

Download full-text

Full-text

Available from: Ajiroghene Thomas
  • Source
    • "CHO cells were grown adherently and maintained in Dulbecco's Modified Eagle's Medium high glucose supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL amphotericin, and 2 mM L-glutamine [17]. HEK 293 cells were grown adherently and maintained in the same grow media of CHO with 1 mM sodium pyruvate [18]. All cell lines were cultured at 37°C and aerated with 5% CO2 : 95% O2. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The anticancer activity of isofuranodiene, extracted from Smyrnium olusatrum, was evaluated in human breast adenocarcinomas MDA-MB 231 and BT 474, and Caucasian prostate adenocarcinoma PC 3 cell lines by MTS assay. MTS assay showed a dose-dependent growth inhibition in the tumor cell lines after isofuranodiene treatment. The best antiproliferative activity of the isofuranodiene was found on PC 3 cells with an IC50 value of 29 μ M, which was slightly less than the inhibition against the two breast adenocarcinoma cell lines with IC50 values of 59 and 55 μ M on MDA-MB 231 and BT 474, respectively. Hoechst 33258 assay was performed in order to study the growth inhibition mechanism in prostate cancer cell line; the results indicate that isofuranodiene induces apoptosis. Overall, the understudy compound has a good anticancer activity especially towards the PC 3. On the contrary, it is less active on Chinese hamster ovary cells (CHO) and human embryonic kidney (HEK 293) appearing as a good candidate as a potential natural anticancer drug with low side effects.
    Full-text · Article · May 2014 · The Scientific World Journal
  • Source
    • "An orphan GPCR, GPR17, which is located phylogenetically at an intermediate position between P2Y and cysteinyl leukotriene receptors (CysLTRs), was reported to be activated by cysteinyl leukotrienes, uracil nucleotides, and nucleotide sugars (UDP, UDP-glucose, and UDP-galactose) and promote mobilization of intracellular Ca 21 and inhibition of cAMP accumulation (Ciana et al., 2006). Although multiple follow-up studies consistent with this original report have been published by the same group (Parravicini et al., 2008; Pugliese et al., 2009; Buccioni et al., 2011; Daniele et al., 2011; Coppi et al., 2013), Benned-Jensen and Rosenkilde (2010) reported that UDP and UDP-glucose, but not cysteinyl leukotrienes, promoted GPR17-dependent guanosine 59-O-(3-[ 35 S]thio)triphosphate binding, and Maekawa et al. (2009) concluded that GPR17 was not activated by any of these presumed agonists but instead was a negative regulator of CysLTR1. Given these disparate results, we have reexamined the potential activation of GPR17 in a broad range of cell types that either stably or transiently express this GPCR. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The orphan receptor GPR17 has been reported to be activated by UDP, UDP-sugars and cysteinyl leukotrienes, and coupled to intracellular Ca(2+) mobilization and inhibition of cyclic AMP accumulation, but other studies have reported either a different agonist profile or lack of agonist activity altogether. To determine if GPR17 is activated by uracil nucleotides and leukotrienes, the HA-tagged receptor was expressed in five different cell lines and the signaling properties of the receptor were investigated. In C6, 1321N1 or CHO cells stably expressing GPR17, UDP, UDP-glucose, UDP-galactose, and cysteinyl-leukotriene C4 (LTC4) all failed to promote inhibition of forskolin-stimulated cyclic AMP accumulation, whereas both UDP and UDP-glucose promoted marked inhibition (>80%) of forskolin-stimulated cyclic AMP accumulation in C6 and CHO cells expressing the P2Y14 receptor. Likewise, none of these compounds promoted accumulation of inositol phosphates in COS-7 or HEK293 cells transiently transfected with GPR17 alone or co-transfected with Gαq/i5, which links Gi-coupled receptors to the Gq-regulated phospholipase C signaling pathway, or PLCε, which is activated by the Gα12/13 signaling pathway. Moreover, none of these compounds promoted internalization of GPR17 in 1321N1-GPR17 cells. Consistent with previous reports, co-expression experiments of GPR17 with CysLTR1 suggested that GPR17 acts as a negative regulator of CysLTR1. Taken together, these data suggest that UDP, UDP-glucose, UDP-galactose and LTC4 are not the cognate ligands of GPR17.
    Preview · Article · Aug 2013 · Journal of Pharmacology and Experimental Therapeutics
  • [Show abstract] [Hide abstract]
    ABSTRACT: Introduction: G protein-coupled receptors (GPCRs) are the largest and most versatile group of cytomembrane receptors, comprising of approximately 300 non-sensory and druggable members. Traditional GPCR drug screening is based on radiometric competition binding assays, which are expensive and hazardous to human health. Furthermore, the paradox of high investment and low output, in terms of new drugs, highlights the need for more efficient and effective drug screening methods. Areas covered: This review summarizes non-radioactive assays assessing the ligand-receptor binding including: the fluorescence polarization assay, the TR-FRET assay and the surface plasmon resonance assay. It also looks at non-radioactive assays that assess receptor activation and signaling including: second messenger-based assays and β-arrestin recruitment-based assays. This review also looks at assays based on cellular phenotypic change. Expert opinion: GPCR signaling pathways look to be more complicated than previously thought. The existence of receptor allosteric sites and multireceptor downstream effectors restricts the traditional assay methods. The emergence of novel drug screening methods such as those for assessing β-arrestin recruitment and cellular phenotypic change may provide us with improved drug screening efficiency and effect.
    No preview · Article · Jun 2012 · Expert Opinion on Drug Discovery
Show more