MAP kinase activation and G_s signaling parameters of wild type TAAR5 and chimeric receptors.

<p><b>(A)</b> HEK293 cells expressing mouse or human TAAR5 or chimeric receptors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117774#pone.0117774.t002" target="_blank">Table 2</a> for details) were stimulated with 100 μM DMEA. The cAMP accumulation was measured by competitive cAMP assay based on AlphaScreen technology. Results are depicted as either fold over basal mock or fold over DMEA stimulated mock transfection. Data are shown as mean ± SEM from n ≥ 3 independent experiments with three or more replicates. Statistical analyses were carried out with an unpaired two-tailed Welch-corrected t-test; ***p ≤ 0.001, compared to the respective basal activity. <b>(B)</b> MAP kinase activation was reported by luciferase activity in a luciferase reporter gene assay (SRE-luc). HEK293 cells were co-transfected with a reporter construct containing a serum response element and the firefly luciferase reporter gene, and the different receptor constructs. Cells were stimulated with 10 μM 3-T₁AM and SRE-luc levels were determined. Results are presented as mean ± SEM as either fold over basal mock transfection for basal value or fold over 3-T₁AM-stimulated mock. An unpaired two-tailed Welsh-corrected t-test was performed for statistical analyses; *p ≤ 0.05.</p

Signaling pathways, basal activity and stimulation with 3-T₁AM at mouse and human TAAR5.

<p>Signaling pathways, basal activity and stimulation with 3-T₁AM at mouse and human TAAR5.</p

Signaling parameters of human and murine TAAR5 after treatment with 3-T₁AM.

<p><b>(A)</b> HEK293 cells expressing human TAAR1 were stimulated with 10 μM 3-T₁AM. For G_s signal determination cAMP accumulation was measured. Results are depicted as fold over basal mock or fold over 3-T₁AM-stimulated mock. Data is shown as mean ± SEM from n ≥ 3 independent experiments with 3 or more replicates. 3-T₁AM is a potent agonist for hTAAR1 (**p < 0.01). Statistical analysis was carried out with an unpaired two-tailed Welch-corrected t-test. <b>(B)</b> HEK293 cells transiently expressing hTAAR5 or mTaar5 were stimulated with 10 μM 3-T₁AM and IP3-luc levels were determined. Results are presented as either fold over basal mock transfection for basal value or fold over 3-T₁AM stimulated mock. An unpaired two-tailed Welsh-corrected t-test was used for statistical analyses, **p ≤ 0.01. Data are obtained from 3 to 6 independent experiments measured in at least triplicates and are shown as mean ± SEM. <b>(C)</b> Human TAAR5 was stimulated with 3-T₁AM concentrations ranging from 1 nM to 100 μM. The concentration-dependent IP3-luc signaling curve indicated the inverse agonism of 3-T₁AM at hTAAR5 with an EC₅₀ value of 4.4 ± 0.9 μM. <b>(D)</b> MAP kinase activation was determined by luciferase activity in a luciferase reporter gene assay (SRE-luc). HEK293 cells were co-transfected with a reporter construct containing a serum response element linked to the firefly luciferase reporter gene and in combination with the different receptor constructs, respectively. Cells were stimulated with 10 μM 3-T₁AM and SRE-luc levels were determined. Results are presented as mean ± SEM as either fold over basal mock transfection for basal value or fold over 3-T₁AM-stimulated mock. An unpaired two-tailed Welsh-corrected t-test was performed for statistical analyses; *p ≤ 0.05. <b>(E)</b> Cell surface expression studies of hTAAR5 were conducted in COS-7 cells for 6 hours after stimulation with or without 10 μM 3-T₁AM using an ELISA. Results are depicted as mean ± SEM obtained from 3 independent assays measured in 4 replicates. Data are presented as fold over basal hTAAR5. An unpaired two-tailed t-test with Welch-correction was performed.</p

Expression of mTaar1 and mTaar5 in various murine brain regions.

<p>Transcript expression studies were analyzed by <i>in situ</i> hybridization using a LNA (locked nucleic acid) probe. C57BL/6 mouse brains were sectioned and treated with the corresponding LNA probes. Signals were visualized by an avidin-biotin complex using DY-light 488 streptavidin <b>(A-F)</b> or DAB (3,3’-diaminobenzidin) staining <b>(G-M)</b>. III = third ventricle; OT = optical tract; DY-light 488 streptavidin labeled samples are shown with a 40-fold, DAB stained sections are depicted with a 20-fold magnification. Bar scale in (B) equals 100 μm, bar scale in (K) equals 200 μm. <b>(A-F)</b>: mTaar5 expression; <b>(A), (C)</b> and <b>(E)</b> represent negative controls using a scrambled LNA probe showing homogenous staining. <b>(B), (D)</b> and <b>(F)</b> show expression of mTaar5 in arcuate nucleus (ARC), in ventromedial hypothalamus (VMH) and amygdala, respectively. <b>(G-M)</b>: mTaar1 expression, the brain regions of interest are highlighted by circles. <b>(G)</b>, <b>(I)</b> and (<b>L)</b> represent negative controls using a scrambled LNA probe. <b>(K)</b> and <b>(M)</b> show expression of mTaar1 in mice brains in VMH and amygdala, respectively. <b>(H)</b> No expression could be detected in the ARC.</p

Structural homology model of hTAAR5 with highlighted amino acid positions that were substituted to design chimeric human-murine receptors.

<p>The design of a structural homology model of hTAAR5 was already reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117774#pone.0117774.ref043" target="_blank">43</a>]. We used this 3D information to visualize the amino acids differing between human and mice TAAR5 in accordance to observed differences in the sequence of both species (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117774#pone.0117774.g004" target="_blank">Fig. 4</a>) with a focus on residues that are in spatial proximity to the extracellular ligand binding part or the intracellular effector binding region. The human wild type amino acids (shown by atom spheres) and the equivalent residues in mouse are provided as mutations in the labels. H1–7 = seven-transmembrane helices 1–7; H8 = eighth intracellular helix; ECL = extracellular loop, ICL = intracellular loop, Ntt = N-terminal tail; Ctt = C-terminal tail.</p

Functional characterization of mouse and human TAAR5 in cAMP and IP₃ assays.

<p><b>(A)</b> Cell surface expression was tested using an ELISA. Mouse and human TAAR5 were N-terminally HA-tagged and transiently transfected with COS-7 cells. Results are depicted as mean ± SEM obtained from 3 independent assays measured in 4 replicates. Data are presented as fold over mock transfection. An unpaired two-tailed t-test was performed which showed no significant difference of cell surface expression between both receptor orthologs. <b>(B)</b> HEK293 cells expressing mouse or human TAAR5 were stimulated with 100 μM DMEA. The cAMP accumulation was measured by competitive cAMP assay based on AlphaScreen technology. Results are depicted as either fold over basal mock or fold over DMEA stimulated mock transfection. Data are shown as mean ± SEM from n ≥ 3 independent experiments with 3 or more replicates. Statistical analyses were carried out with an unpaired two-tailed Welch-corrected t-test; ***p ≤ 0.001, compared to the respective basal activity. <b>(C)</b> HEK293 cells transiently expressing hTAAR5 or mTaar5 were incubated with or without 500 nM pertussis toxin (PTX), 20 hours prior to measurement. Supplement-free MEM was added to the untreated cell. IP3-luc levels were determined without (basal) and with PTX treatment. Results are presented as relative light units (RLU). Data were obtained from 3 to 6 independent experiments measured in triplicates and are shown as mean ± SEM. An unpaired two-tailed Welsh-corrected t-test was used for statistical analyses; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Human TAAR5 shows elevated basal IP3-luc activity, which was maintained after treatment with PTX pointing to a basal G_q/11 activity. Basal IP3-luc activity of mTaar5 was not significantly influenced by PTX treatment.</p