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The asthma candidate gene NPSR1 mediates isoform specific downstream signalling.

Pietras CO, Vendelin J, Anedda F, Bruce S, Adner M, Sundman L, Pulkkinen V, Alenius H, D'Amato M, Söderhäll C, Kere J - BMC Pulm Med (2011)

Bottom Line: The results were verified by NPS concentration-response and time series analysis using qRT-PCR, cAMP and Ca²⁺ assays, and cAMP/PKA, MAPK/JNK and MAPK/ERK pathway specific reporter assays.NPSR1-B signalled through the same pathways and regulated the same genes as NPSR1-A, but NPSR1-B yielded lower induction on effector genes than NPSR1-A, with one notable exception, CD69, a marker of regulatory T cells.We conclude that NPSR1-B is regulating essentially identical set of genes as NPSR1-A, with few, but possibly important exceptions, and that NPSR1-A induces stronger signalling effects than NPSR1-B.

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Affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Background: Neuropeptide S Receptor 1 (NPSR1, GPRA, GPR154) was first identified as an asthma candidate gene through positional cloning and has since been replicated as an asthma and allergy susceptibility gene in several independent association studies. In humans, NPSR1 encodes two G protein-coupled receptor variants, NPSR1-A and NPSR1-B, with unique intracellular C-termini. Both isoforms show distinct expression pattern in asthmatic airways. Although NPSR1-A has been extensively studied, functional differences and properties of NPSR1-B have not yet been clearly examined. Our objective was to investigate downstream signalling properties of NPSR1-B and functional differences between NPSR1-A and NPSR1-B.

Methods: HEK-293 cells transiently overexpressing NPSR1-A or NPSR1-B were stimulated with the ligand neuropeptide S (NPS) and downstream signalling effects were monitored by genome-scale affymetrix expression-arrays. The results were verified by NPS concentration-response and time series analysis using qRT-PCR, cAMP and Ca²⁺ assays, and cAMP/PKA, MAPK/JNK and MAPK/ERK pathway specific reporter assays.

Results: NPSR1-B signalled through the same pathways and regulated the same genes as NPSR1-A, but NPSR1-B yielded lower induction on effector genes than NPSR1-A, with one notable exception, CD69, a marker of regulatory T cells.

Conclusions: We conclude that NPSR1-B is regulating essentially identical set of genes as NPSR1-A, with few, but possibly important exceptions, and that NPSR1-A induces stronger signalling effects than NPSR1-B. Our findings suggest an isoform-specific link to pathogenetic processes in asthma and allergy.

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Phosphorylation site-directed mutagenesis of NPSR1-A and NPSR1-B C-termini. Relative CGA mRNA expression after 1, 6 and 24 h NPS stimulation of HEK-293 cells overexpressing NPSR1-A or -B containing phosphorylation site mutations in the C-termini. From left to right: NPSR1-A,-AΔ5p, -AΔ3p, -AΔ2p, NPSRI-B and -BΔ2p. Expression shown as means relative to an NPS stimulated empty vector control ± SEM.
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Figure 7: Phosphorylation site-directed mutagenesis of NPSR1-A and NPSR1-B C-termini. Relative CGA mRNA expression after 1, 6 and 24 h NPS stimulation of HEK-293 cells overexpressing NPSR1-A or -B containing phosphorylation site mutations in the C-termini. From left to right: NPSR1-A,-AΔ5p, -AΔ3p, -AΔ2p, NPSRI-B and -BΔ2p. Expression shown as means relative to an NPS stimulated empty vector control ± SEM.

Mentions: To dissect the mechanisms of different regulatory effects between the NPSR1-A and -B isoforms, we performed phosphorylation site-directed mutagenesis. The NPSR1-A and -B isoforms possess distinct C-termini which, together with the third intracellular loop, contain phosphorylation sites potentially important for arrestin docking [18]. Phosphorylation occurs predominantly on serine (S) and threonine (T) residues [32] and, as illustrated in Figure 1b, NPSR1-A carries five unique C-terminal phosphorylation sites, whereas NPSR1-B only two. We generated NPSR1-A constructs with all five unique phosphorylation sites in the C-terminal mutated to alanine (AΔ5p), three sites mutated (AΔ3p) and two sites mutated (AΔ2p) and NPSR1-B constructs with the only two unique sites mutated (BΔ2p) (Additional file 2, Figure S1). HEK-293 cells were transiently transfected with the mutant constructs or the wild type pCMV-NPSR1-A or NPSR1-B, stimulated with NPS for 6 h and the downstream expression of a subset of genes were measured with qRT-PCR. The results showed that the removal of phosphorylation sites did not have any effect on downstream gene expression (representative gene shown in Figure 7), suggesting that the significant differential effect between NPSR1-A and -B involves mechanism(s) distinct from simple phosphorylation differences.


The asthma candidate gene NPSR1 mediates isoform specific downstream signalling.

Pietras CO, Vendelin J, Anedda F, Bruce S, Adner M, Sundman L, Pulkkinen V, Alenius H, D'Amato M, Söderhäll C, Kere J - BMC Pulm Med (2011)

Phosphorylation site-directed mutagenesis of NPSR1-A and NPSR1-B C-termini. Relative CGA mRNA expression after 1, 6 and 24 h NPS stimulation of HEK-293 cells overexpressing NPSR1-A or -B containing phosphorylation site mutations in the C-termini. From left to right: NPSR1-A,-AΔ5p, -AΔ3p, -AΔ2p, NPSRI-B and -BΔ2p. Expression shown as means relative to an NPS stimulated empty vector control ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3142248&req=5

Figure 7: Phosphorylation site-directed mutagenesis of NPSR1-A and NPSR1-B C-termini. Relative CGA mRNA expression after 1, 6 and 24 h NPS stimulation of HEK-293 cells overexpressing NPSR1-A or -B containing phosphorylation site mutations in the C-termini. From left to right: NPSR1-A,-AΔ5p, -AΔ3p, -AΔ2p, NPSRI-B and -BΔ2p. Expression shown as means relative to an NPS stimulated empty vector control ± SEM.
Mentions: To dissect the mechanisms of different regulatory effects between the NPSR1-A and -B isoforms, we performed phosphorylation site-directed mutagenesis. The NPSR1-A and -B isoforms possess distinct C-termini which, together with the third intracellular loop, contain phosphorylation sites potentially important for arrestin docking [18]. Phosphorylation occurs predominantly on serine (S) and threonine (T) residues [32] and, as illustrated in Figure 1b, NPSR1-A carries five unique C-terminal phosphorylation sites, whereas NPSR1-B only two. We generated NPSR1-A constructs with all five unique phosphorylation sites in the C-terminal mutated to alanine (AΔ5p), three sites mutated (AΔ3p) and two sites mutated (AΔ2p) and NPSR1-B constructs with the only two unique sites mutated (BΔ2p) (Additional file 2, Figure S1). HEK-293 cells were transiently transfected with the mutant constructs or the wild type pCMV-NPSR1-A or NPSR1-B, stimulated with NPS for 6 h and the downstream expression of a subset of genes were measured with qRT-PCR. The results showed that the removal of phosphorylation sites did not have any effect on downstream gene expression (representative gene shown in Figure 7), suggesting that the significant differential effect between NPSR1-A and -B involves mechanism(s) distinct from simple phosphorylation differences.

Bottom Line: The results were verified by NPS concentration-response and time series analysis using qRT-PCR, cAMP and Ca²⁺ assays, and cAMP/PKA, MAPK/JNK and MAPK/ERK pathway specific reporter assays.NPSR1-B signalled through the same pathways and regulated the same genes as NPSR1-A, but NPSR1-B yielded lower induction on effector genes than NPSR1-A, with one notable exception, CD69, a marker of regulatory T cells.We conclude that NPSR1-B is regulating essentially identical set of genes as NPSR1-A, with few, but possibly important exceptions, and that NPSR1-A induces stronger signalling effects than NPSR1-B.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Background: Neuropeptide S Receptor 1 (NPSR1, GPRA, GPR154) was first identified as an asthma candidate gene through positional cloning and has since been replicated as an asthma and allergy susceptibility gene in several independent association studies. In humans, NPSR1 encodes two G protein-coupled receptor variants, NPSR1-A and NPSR1-B, with unique intracellular C-termini. Both isoforms show distinct expression pattern in asthmatic airways. Although NPSR1-A has been extensively studied, functional differences and properties of NPSR1-B have not yet been clearly examined. Our objective was to investigate downstream signalling properties of NPSR1-B and functional differences between NPSR1-A and NPSR1-B.

Methods: HEK-293 cells transiently overexpressing NPSR1-A or NPSR1-B were stimulated with the ligand neuropeptide S (NPS) and downstream signalling effects were monitored by genome-scale affymetrix expression-arrays. The results were verified by NPS concentration-response and time series analysis using qRT-PCR, cAMP and Ca²⁺ assays, and cAMP/PKA, MAPK/JNK and MAPK/ERK pathway specific reporter assays.

Results: NPSR1-B signalled through the same pathways and regulated the same genes as NPSR1-A, but NPSR1-B yielded lower induction on effector genes than NPSR1-A, with one notable exception, CD69, a marker of regulatory T cells.

Conclusions: We conclude that NPSR1-B is regulating essentially identical set of genes as NPSR1-A, with few, but possibly important exceptions, and that NPSR1-A induces stronger signalling effects than NPSR1-B. Our findings suggest an isoform-specific link to pathogenetic processes in asthma and allergy.

Show MeSH
Related in: MedlinePlus