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: 3.51). 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, Jul 12, 2015
0 Followers
 · 
148 Views
  • [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.
    Expert Opinion on Drug Discovery 06/2012; 7(9):791-806. DOI:10.1517/17460441.2012.699036
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Unveiling the mechanisms participating in the damage and repair of traumatic brain injury (TBI) is fundamental to develop new therapies. The P2Y-like GPR17 receptor has recently emerged as a sensor of damage and a key actor in lesion remodeling/repair in the rodent brain, but its role in humans is totally unknown. Here, we characterized GPR17 expression in brain specimens from seven intensive care unit TBI patients undergoing neurosurgery for contusion removal and from 28 autoptic TBI cases (and 10 control subjects of matched age and gender) of two university hospitals. In both neurosurgery and autoptic samples, GPR17 expression was strong inside the contused core and progressively declined distally according to a spatio-temporal gradient. Inside and around the core, GPR17 labeled dying neurons, reactive astrocytes, and activated microglia/macrophages. In peri-contused parenchyma, GPR17 decorated oligodendrocyte precursor cells (OPCs) some of which had proliferated, indicating re-myelination attempts. In autoptic cases, GPR17 expression positively correlated with death for intracranial complications and negatively correlated with patients' post-traumatic survival. Data indicate lesion-specific sequential involvement of GPR17 in the (a) death of irreversibly damaged neurons, (b) activation of microglia/macrophages remodeling the lesion, and (c) activation/proliferation of multipotent parenchymal progenitors (both reactive astrocytes and OPCs) starting repair processes. Data validate GPR17 as a target for neurorepair and are particularly relevant to setting up new therapies for TBI patients.
    Purinergic Signalling 06/2013; 9(3). DOI:10.1007/s11302-013-9366-3
  • [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.
    Journal of Pharmacology and Experimental Therapeutics 08/2013; 347(1). DOI:10.1124/jpet.113.207647
Show more