Sequence: G protein-coupled receptor for asthma susceptibility isoform A [Homo sapiens]

Gene:NPSR1     Conserved Domain     Related Protein 3D Structures

AID	Name	Type	Probe	Comment
624005	Extended Characterization of Antagonists of the Neuropeptide S Receptor: Summary	summary		EC Summary AID
1461	qHTS Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transduction	confirmatory
1464	Quantitative High-Throughput Screen for Antagonists of the Neuropeptide S Receptor: Summary	summary	2
434931	Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Robust Characterization of Calcium Signal Transduction	confirmatory
2568	Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transduction, SAR for Probe	confirmatory
434936	Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: Robust Characterization of cAMP Signal Transduction	confirmatory
624054	Robust Characterization of cAMP Signal Transduction for Antagonists of the Neuropeptide S Receptor: SAR	other

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]

MLPCN Grant: X01-DA026210-01
Assay Submitter (PI): Heilig, Markus Alexander

NCGC Assay Overview:

Neuropeptide S receptor (NPSR), previously known as GPR154, is a recently de-orphanized G protein coupled receptor. Its endogenous ligand is the 20 amino acids peptide Neuropeptide S (NPS). Activation of NPSR induces transient increases in intracellular calcium and cAMP, suggesting coupling of this receptor to both Gs and Gq G proteins. NPS and its receptor are found in various tissues. Specifically they are highly expressed in brain areas that have been implicated in modulation of arousal, stress and anxiety. Central administration of NPS in mice produces an unusual profile of activity by inducing wakefulness and arousal, while at the same time suppressing anxiety. Therefore, NPSR may represent a novel drug target for the treatment of sleep and anxiety disorders.

As one method of confirming the specific activity of compounds active in the primary screen (AID 1461), we developed an assay to measure the displacement of radio-labeled native ligand, NPS, from its receptor in vitro. The lack of displacement does not necessarily indicate that a compound is non-specific for the NPS receptor, rather, it indicates that the binding site is unlikely to be the same as the native ligand.

The assay was carried out as described (Xu et al. (2004) Neuron, 43: 487-497) with minor modification.

Y10-NPS labeled with 125I was bought from NEN Perkin Elmer (Boston, MA). CHO cells stably expressing human NPSR were seeded into 24-well plates and cultured until reaching 90-95% confluency. Cells were washed with 1ml PBS once and then incubated with radioligand with or without compounds or in DMEM medium containing 0.1% bovine serum albumin at 20C for 1.5 hr. Increasing concentrations of compounds or unlabeled human NPS were used to compete with 0.15 nM [125I] Y10-NPS. Nonspecific binding was determined in the presence of 1 uM unlabeled human NPS. Cells were washed twice with cold PBS and lysed with 1 N NaOH. Bound radioactivity was counted in a liquid scintillation counter.

Compounds displacing radio-labeled ligand were scored 50. Other compounds are inconclusive and are scored 10.

Grant Number: X01-DA026210-01