Multiple polymorphisms affect expression and function of the neuropeptide S receptor (NPSR1)

Abstract

Background: neuropeptide S (NPS) and its receptor NPSR1 act along the hypothalamic-pituitary-adrenal axis to modulate anxiety, fear responses, nociception and inflammation. The importance of the NPS-NPSR1 signaling pathway is highlighted by the observation that, in humans, NPSR1 polymorphism associates with asthma, inflammatory bowel disease, rheumatoid arthritis, panic disorders, and intermediate phenotypes of functional gastrointestinal disorders. Because of the genetic complexity at the NPSR1 locus, however, true causative variations remain to be identified, together with their specific effects on receptor expression or function. To gain insight into the mechanisms leading to NPSR1 disease-predisposing effects, we performed a thorough functional characterization of all NPSR1 promoter and coding SNPs commonly occurring in Caucasians (minor allele frequency >0.02).

Principal findings: we identified one promoter SNP (rs2530547 [-103]) that significantly affects luciferase expression in gene reporter assays and NPSR1 mRNA levels in human leukocytes. We also detected quantitative differences in NPS-induced genome-wide transcriptional profiles and CRE-dependent luciferase activities associated with three NPSR1 non-synonymous SNPs (rs324981 [Ile107Asn], rs34705969 [Cys197Phe], rs727162 [Arg241Ser]), with a coding variant exhibiting a loss-of-function phenotype (197Phe). Potential mechanistic explanations were sought with molecular modelling and bioinformatics, and a pilot study of 2230 IBD cases and controls provided initial support to the hypothesis that different cis-combinations of these functional SNPs variably affect disease risk.

Significance: these findings represent a first step to decipher NPSR1 locus complexity and its impact on several human conditions NPS antagonists have been recently described, and our results are of potential pharmacogenetic relevance.

Figure 1. NPSR1 polymorphisms characterized in this study, and vector constructs used for their functional analysis.

Top: NPSR1 common (minor allele frequency [MAF] >0.02) promoter and coding SNPs and their location in the gene region. Left: Chromosomal position, MAF, and functional effect of NPSR1 SNPs (minor alleles in lower case). Right: Vectors used to functionally characterize NPSR1 promoter (top) and coding (bottom) SNPs. For coding polymorphisms, a Myc-tagged version of each construct has also been produced for the experiments of immunofluorescence and FACS analysis.

Figure 2. Effect of NPSR1 promoter polymorphism on gene transcription.

A) Colo205 cells were transiently transfected with NPSR1 promoter constructs as indicated, in triplicate experiments. Twenty-four hours after transfection luciferase activity was measured and expressed as fold changes (+ SD) relative to the reference construct pNPSR1P. The substitution of a cytosine (C) with a thymidine (T) at position −103 (SNP rs2530547) resulted in significantly lower levels of luciferase activity (pNPSR1P-103T vs pNPSR1P, Mann-Whitney U test P = 0.004). B) Blood levels of NPSR1 mRNA were detected with TaqMan NPSR1-specific Real-Time PCR assay in 42 healthy volunteers, and expressed in arbitrary units according to genotype at position -103 of NPSR1 promoter (SNP rs2530547). Homozygosity for the T allele at this locus correlated with lower levels of NPSR1 mRNA (Mann-Whitney U test P = 0.009).

Figure 3. Membrane expression of NPSR1 coding variants.

Top: HEK293 cells were transiently transfected with Myc-tagged NPSR1 expression vectors corresponding to different coding variants as indicated, and stained with anti-Myc antibody (red) and Hoechst 33258 (blue). Bottom: FACS analysis of cell surface expression of NPSR1 coding variants HEK293 cells were co-transfected with GFP and either an empty vector (ctrl) or vectors carrying Myc-tagged cDNAs for NPSR1 and all coding variants as indicated. After gating for GFP, anti-Myc mean fluorescence intensity (MIF) was measured for each sample, and the results obtained in three independent experiments are reported in the graph. In these conditions, the expression of the pNPSR1-107Asn variant was significantly lower (t test) than that of pNPSR1, taken as reference for this series of experiments.

Figure 4. Pharmacological activity of NPSR1 coding variants.

Dose-response curves of NPS stimulation, in HEK293 cells transiently co-transfected with a CRE-luc plasmid and NPSR1 coding variants in triplicate, as indicated. One representative experiment is reported, where CRE-driven luciferase activity is expressed in mean arbitrary units ± SEM.

Figure 5. Molecular model of NPSR1.

A) The molecular model of NSPR1 indicates that residues Ile107, Cys197 and Agr 241 are localized at each end of the transmembrane NSPR1 protein, while residue Cys197 is within the presumptive NPS-binding site. The model (side view) is oriented with the extracellular matrix and the cytosolic compartment at the top and at the bottom, respectively. Each of the seven helices that compose the NPSR1 transmembrane core is indicated with a different color and annotated with H. The side chains of the four different residues are displayed as spheres. B) View of the molecular model of NPSR1 seen from the extracyoplasmic domain. Residues Ile107, Cys197 and Cys121 are indicated as sticks. The side chains of Cys197 and Cys121 form a disulfide bridge within the NPS-binding site.

Figure 6. Functional variability in NPSR1 receptor signalling.

Based on the results obtained in this study, allelic differences in mRNA expression and protein signalling capacity exist, respectively, at SNPs corresponding to promoter position −103 and amino acid residues 107, 197 and 241. This is graphically represented by vertical arrows in the figure, where more arrows indicate higher expression or stronger signalling, respectively, for promoter and coding SNPs. In theory, this results in 16 potential cis combinations of NPSR1 functional alleles into haplotypes, and 136 different pairs of such haplotypes from 2 chromosomes, establishing a very large gradient of NPSR1 functional variation ranging from low/absent to very high signalling.