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Gen Comp Endocrinol
2020 Dec 01;299:113586. doi: 10.1016/j.ygcen.2020.113586.
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Identification and signaling characterization of four urotensin II receptor subtypes in the western clawed frog, Xenopus tropicalis.
Konno N
,
Takano M
,
Miura K
,
Miyazato M
,
Nakamachi T
,
Matsuda K
,
Kaiya H
.
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Urotensin II (UII) is involved, via the UII receptor (UTR), in many physiological and pathological processes, including vasoconstriction, locomotion, osmoregulation, immune response, and metabolic syndrome. In silico studies have revealed the presence of four or five distinct UTR (UTR1-UTR5) gene sequences in nonmammalian vertebrates. However, the functionality of these receptor subtypes and their associations to signaling pathways are unclear. In this study, full-length cDNAs encoding four distinct UTR subtypes (UTR1, UTR3, UTR4, and UTR5) were isolated from the western clawed frog (Xenopus tropicalis). In functional analyses, homologous Xenopus UII stimulation of cells expressing UTR1 or UTR5 induced intracellular calcoum mobilization and phosphorylation of extracellular signal-regulated kinase 1/2. Cells expressing UTR3 or UTR4 did not show this response. Furthermore, UII induced the phosphorylation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) through the UII-UTR1/5 system. However, intracellular cAMP accumulation was not observed, suggesting that UII-induced CREB phosphorylation is caused by a signaling pathway different from that involving Gs protein. In contrast, the administration of UII to cells increased the phosphorylation of guanine nucleotide exchange factor-H1 (GEF-H1) and myosin light chain 2 (MLC2) in all UTR subtypes. These results define four distinct UTR functional subtypes and are consistent with the molecular evolution of UTR subtypes in vertebrates. Further understanding of signaling properties associated with UTR subtypes may help in clarifying the functional roles associated with UII-UTR interactions in nonmammalian vertebrates.
Fig. 1. Deduced amino acid sequences of urotensin II receptor (UTR) subtypes identified in Xenopus tropicalis. Deduced sequences were aligned using the ClustalW algorithm (asterisks indicate identical amino acid residues; open boxes predicted transmembrane regions; black boxes, putative N-glycosylation sites; gray characters, putative phosphorylation sites; gray boxes, LxxxD motif (where x represents any amino acid); hash, D/ERY motif; closed circles, conserved cysteine residues; and black triangles, NPxxY motif).
Fig. 2. Phylogenetic analysis of urotensin II receptor (UTR) subtypes in vertebrates. The phylogenetic tree was constructed by MEGA6 using the maximum-likelihood method. Data were resampled with 1000 bootstrap iterations. The accession numbers of the sequences are provided in Table S1.
Fig. 3. Relative mRNA expression levels for urotensin II receptor (UTR) subtypes in various tissues of Xenopus tropicalis. mRNA expression levels of single UTR subtypes are represented as ratios to the mRNA level of the housekeeping gene RPS13. Values are estimates for samples from three frogs. A–D, relative mRNA expression level of UTR subtypes in male and female frogs.
Fig. 4. Dose–response relationships of the urotensin II (UII)- or urotensin II-related peptide (URP)-mediated Ca2+ mobilization in Chinese hamster ovary (CHO) cells expressing single UTR subtypes. CHO cells transfected with single urotensin II receptor (UTR) subtypes were incubated with different concentrations (0.1–1000 nM) of human UII, Xenopus UII, URP, or somatostatin. Transfected cells were loaded with Fluo-4 Ca2+-sensitive dye and then stimulated with different concentrations of ligands. Fluorescence intensities for Ca2+ mobilization were determined using a fluorometric imaging plate reader (FLIPR) (A–D). Cells pretreated with the UTR antagonist (urantide) were stimulated with the indicated concentrations of ligands (E and F). The data are the means of duplicate wells.
Fig. 5. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) by urotensin II (UII) stimulation in the cells expressing single urotensin II receptor (UTR) subtypes. (A) Cells transfected with single UTR subtype genes were stimulated with UII (100 nM) for 30 min. Cell extracts were western blotted using antibodies for phosphorylated and total ERK1/2 protein. The indicated blot is representative of three independent experiments. (B) The phosphorylation levels of ERK1/2 were normalized to total ERK1/2 levels. Values are expressed as fold increase vs. unstimulated cells (control) and are presented as the mean ± SEM of three independent experiments. *p < 0.05 vs. control (B and E, Welch’s test; C, D, and F, unpaired Student’s t-test).
Fig. 6. Urotensin II (UII)-stimulated luciferase activities in HEK293T cells transfected with single urotensin II receptor (UTR) subtypes and CRE-luciferase reporter vector. Cells were stimulated with UII of the indicated doses of UII for 2 h. UII-stimulated luciferase activities in the cells expressing UTR1 (A) or UTR5 (D) were significantly increased compared with those of unstimulated cells expressing UTR1 or UTR5. Cells expressing UTR3 (B) or UTR4 (C) showed no response to UII stimulation. Values are expressed as fold increase vs. unstimulated cells (control) and are presented as the mean ± SEM of three independent experiments. **p < 0.01, *p < 0.05 vs. control (one-way analysis of variance using Dunnett’s test). (E) Cells transfected with single UTR subtypes were stimulated with UII (100 nM) for 30 min. Cell extracts were western blotted using antibodies for phosphorylated and total CREB protein. The indicated blot is representative of two independent experiments.
Fig. 7. Dose–response relationship of cyclic adenosine monophosphate (cAMP) production in Chinese hamster ovary (CHO) cells expressing urotensin II receptor (UTR) 1, UTR5, or vasotocin type-2 receptor (VT2R). CHO cells transfected with UTR1 (A), UTR5 (B), or VT2R (C) were incubated with different concentrations (0.1–1000 nM) of UII or vasotocin (VT) for 2 h. cAMP concentration was measured using an Alpha-screen cAMP assay kit. Data are means of triplicate wells from one representative of two independent experiments. Different letters in the plots indicate significant differences at p < 0.05 (one-way analysis of variance using Bonferroni’s multiple comparison test).
Fig. 8. Phosphorylation of guanine nucleotide exchange factor H1 (GEF-H1) by urotensin II (UII) stimulation in the cells expressing single urotensin II receptor (UTR) subtypes. (A) Cells transfected with single UTR subtypes were stimulated with UII (100 nM) for 30 min. Cell extracts were western blotted using antibodies for phosphorylated and total GEF-H1 protein. The indicated blot is representative of three independent experiments. (B) Phosphorylation levels of GEF-H1 were normalized to total GEF-H1 levels. Values are expressed as fold increase vs. unstimulated cells (control) and are presented as the mean ± SEM of three independent experiments. **p < 0.01, *p < 0.05 vs. control (Welch’s test).
Fig. 9. Phosphorylation of myosin light chain (MLC) by urotensin II (UII) stimulation in the cells expressing single urotensin II receptor (UTR) subtypes. (A) Cells transfected with single UTR subtypes were stimulated with UII (100 nM) for 30 min. Cell extracts were western blotted using antibodies for phosphorylated and total MLC protein. The indicated blot is representative of three independent experiments. (B) Phosphorylation levels of MLC were normalized to total MLC levels. Values are expressed as fold increase vs. unstimulated cells (control) and are presented as the mean ± SEM of three independent experiments. *p < 0.05 vs. control (Welch’s test).
Fig. 10. Intracellular signaling pathways predicted from functional assays via urotensin II receptor (UTR) subtypes. UTR1 and UTR5 are likely to couple to Gq and G12/13 proteins and are most likely linked to PLC/PKC and GEF-H1/RhoA/ROCK/MLC signaling, similar to mammalian UTR1. cAMP response element-binding protein (CREB) phosphorylation is induced via extracellular signal-regulated kinase and other pathways, except for Gs protein-cAMP. UTR3 and UTR4 are likely linked to GEF-H1/RhoA/ROCK/MLC signaling via the G12/13 protein.
Fig. S1. Deduced amino acid sequences of urotensin II receptor (UTR) subtypes identified in Japanese medaka (Oryzias latipes). Deduced sequences were aligned using the ClustalW algorithm (asterisks indicate identical amino acid residues; open boxes, predicted transmembrane regions; black boxes, putative N-glycosylation sites; gray characters, putative phosphorylation sites; gray boxes, LxxxD motif (where x represents any amino acid); hash, D/ERY motif; closed circles, conserved cysteine residues; and black triangles, NPxxY motif).
Fig. S2. Relative mRNA expression levels for urotensin II receptor (UTR) subtypes in various tissues of O. latipes. mRNA expression levels of single UTR subtypes are represented as ratios to the mRNA level of the housekeeping gene RPS13. Values are estimates for samples from three fish. A–D, relative mRNA expression level of single UTR subtypes in male and female fish.
Fig. S3. Dose–response relationships of urotensin II (UII)- or urotensin II-related peptide (URP)-mediated Ca2+ mobilization in Chinese hamster ovary (CHO) cells expressing single urotensin II receptor (UTR) subtypes of Oryzias latipes. CHO cells transfected with single UTR subtypes were incubated with different doses (0.1–1000 nM) of human UII, Medaka UII, Medaka URP, or somatostatin. Transfected cells were loaded with Fluo-4 Ca2+-sensitive dye and then stimulated with different concentrations of ligands. Fluorescence intensities for Ca2+ mobilization were determined using fluorometric imaging plate reader (FLIPR) (A–D). Cells pretreated with the UTR antagonist urantide were stimulated with the indicated concentrations of ligands (E and F). The data are means of duplicate wells.
Fig. S4. Lamellipodia formation observed after urotensin II (UII) stimulation of urotensin II receptor 1 (UTR1) expressing cells. Living HEK293T cells co-transfected with Xenopus UTR1 and Lifeact-GFP (F-actin marker) were imaged in real-time using time-lapse videomicroscopy for 60 min, and individual frames were recorded before and during exposure to 100 nM UII. Lamellipodia formation (white arrow) was observed within 10 min after the addition of UII.
