title: Importance of extracellular loop one of the neuropeptide S receptor for biogenesis and function.
abstract: Neuropeptide S (NPS) is the endogenous ligand of a formerly orphan G protein-coupled receptor (GPCR). The NPS receptor (NPSR) belongs to the subfamily of peptide GPCRs and is widely expressed in the brain. NPS promotes arousal and induces anxiolytic-like effects after central administration in rodents. Previously, we have reported that the N107I polymorphism in the human NPS receptor results in a gain-of-function characterized by an increase in agonist potency without changing agonist binding affinity. We have extended our findings by investigating pharmacological and biochemical consequences of mutations in the vicinity of position 107. Alanine substitutions were made for D105 and N101, and stable clones were analyzed for agonist-induced changes of intracellular Ca(2+). Receptor protein expression was monitored by Western blot and flow cytometry. The mutation D105A produced receptors that have a approximately 200-fold higher EC(50) despite elevated total receptor protein and surface expression compared to cell lines expressing the parental receptor NPSR-N107. The mutation N101A resulted in slightly reduced agonist potency without affecting the ability of the protein to form functional receptors. Stable NPSR-A101 clones show little expression of the fully glycosylated form. However, NPSR-A101 receptors are expressed on the cell surface and are functional, suggesting that full glycosylation is not required for receptor function. Our studies suggest that N-linked glycosylation is not important for receptor biogenesis or function, and that residue D105 might be critical for receptor binding.
authors.affiliation: Pharmaceutical Sciences, University of California, Irvine, 147 Biological Sciences Administration, Irvine, CA 92697, USA. sdclark@uci.edu
authors.forename: Stewart D
authors.initials: SD
authors.lastname: Clark
authors.forename: Ha T
authors.initials: HT
authors.lastname: Tran
authors.forename: Joanne
authors.initials: J
authors.lastname: Zeng
authors.forename: Rainer K
authors.initials: RK
authors.lastname: Reinscheid
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D023303:Oxazolidinones / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011719:Pyrazines / Q000378:metabolism; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
chemical_list: C529493:3-oxo-1,1-diphenyltetrahydrooxazolo(3,4-a)pyrazine-7-carboxylic acid 4-fluorobenzylamide; C484532:NPSR1 protein, human; D023303:Oxazolidinones; D011719:Pyrazines; D043562:Receptors, G-Protein-Coupled