Analysis of Human TAAR8 and Murine Taar8b Mediated Signaling Pathways and Expression Profile

The thyroid hormone derivative 3-iodothyronamine (3-T1AM) exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T1AM acts as a trace amine-associated receptor 1 (TAAR1) agonist and activates Gs signaling in vitro. Interestingly, 3-T1AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T1AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8). By RT-qPCR and locked-nucleic-acid (LNA) in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of Gs and/or Gi/o signaling. Activation of G-proteins Gq/11 and G12/13 was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T1AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal Gi/o signaling activity, a so far unknown signaling pathway for TAARs

Analysis of variants within the coding sequence and adjacent regions of obesity-related genes

Im Rahmen von genomweiten Assoziationsstudien (GWAS) wurden bisher 32 Genorte identifiziert, für die eine Assoziation mit Adipositas nachgewiesen werden konnte, darunter MC4R (Melanocortin-4-Rezeptor) und GIPR (gastric inhibitory polypeptide receptor). Der MC4R ist an der hypothalamischen Gewichtsregulation und der GIPR an der Insulinsekretion in Reaktion auf orale Aufnahme glukosehaltiger Nahrung beteiligt. Im Rahmen dieser Arbeit wurden genetische Varianten (Single-Nukleotid-Polymorphismen (SNPs) und Mutationen) in der kodierenden sowie angrenzenden Sequenz dieser beiden Gene im Hinblick auf folgende Aspekte untersucht: a) auf ihren Einfluss auf das Auftreten von Adipositas sowie möglichen Komorbiditäten, b) auf die Bedeutung positiver Assoziationssignale, c) auf ihre Verbreitung und d) auf mögliche zukünftige Therapieansätze. Für die von uns analysierten SNPs im Bereich des GIPR konnte keine Assoziation mit Adipositas bestätigt werden, jedoch zeigte sich eine Assoziation mit erhöhten HOMA-IR-Werten (Homeostatic Model Assessment of Insulin Resistance). Für den MC4R konnte ein SNP-Haplotyp identifiziert werden, der mit erhöhtem BMI assoziiert ist. Diese Assoziation ist unabhängig von möglichen MC4R-Mutationen im kodierenden Bereich. Zusätzlich konnten wir erste Hinweise auf einen Haplotyp feststellen, der sowohl zwischen übergewichtigen MC4R-Mutationsträgern und Nichtträgern, als auch zwischen Trägern funktionell relevanter und nicht relevanter Mutationen unterscheidet. Dabei wurde der Einfluss von „cryptic relatedness“ deutlich, da eine größere Anzahl an Studienteilnehmern Träger der häufigen europäischen Doppelmutation [p.Tyr35Stop; c.110 C>T] war. Eine zusätzliche Haplotypanalyse bei den Familien der Doppelmutationsträger und unabhängigen Kontroll-Trios ergab, dass ein „Common-Founder-Effekt“ als sehr wahrscheinlich angenommen werden kann. Da MC4R-Nonsense-Mutationen wie die [p.Tyr35Stop; c.110 C>T] in der Regel mit schwerer, frühmanifester Adipositas einhergehen, wurde schließlich die Möglichkeit eines pharmakologischen Therapieansatzes untersucht. Dabei konnte gezeigt werden, dass der Aminoglykosid-vermittelte Überleseeffekt bei MC4R- Nonsense-Mutationen in Abhängigkeit von verschiedenen Faktoren eine (teilweise) Wiederherstellung der Proteinexpression bzw. Rezeptorfunktion bewirkt. In Zukunft sind weitere Analysen, auch im Hinblick auf polygene Effekte notwendig, um die Zusammenhänge bei genetisch bedingter Adipositas zu verstehen und um weitere Diagnosemethoden und Therapieansätze entwickeln zu können.To date, genome-wide association studies (GWAS) identified 32 genetic loci to be associated with obesity, including MC4R (melanocortin 4 receptor) and GIPR (gastric inhibitory polypeptide receptor). The MC4R plays an important role in the hypothalamic regulation of body weight, and the GIPR is involved in insulin secretion in response to oral glucose uptake. Within the scope of my work, I analyzed genetic variants - single nucleotide polymorphisms (SNPs) and mutations - located in the coding sequences and adjacent regions of MC4R and GIPR with regard to the following aspects: a) their influence on obesity and possible comorbidities, b) the impact of positive association signals, c) their prevalence, and d) possible future treatment approaches. We could not confirm an association between the analyzed SNPs within the GIPR region and obesity. Nevertheless, we detected an association with increased HOMA-IR (homeostatic model assessment of insulin resistance) values. We identified a haplotype of non-coding SNPs near the MC4R to be associated with increased BMI. Possible mutations in the MC4R coding region showed no impact on this association signal. In addition, we found first evidence for a haplotype discriminating between obese mutation carriers and non-carriers, as well as between carriers of functionally relevant and non-relevant mutations. In this context, the influence of “cryptic relatedness” was revealed, since a majority of study participants carried the frequent northern European haplotype [p.Tyr35Stop; c.110 C>T]. An additional haplotype analysis in the families of carriers of the [p.Tyr35Stop; c.110 C>T] mutational haplotype and in independent control trios showed evidence for a common founder effect. Since MC4R nonsense mutations like the [p.Tyr35Stop; c.110 C>T] are, for the most part, associated with severe early onset obesity, we explored possible pharmaceutical treatment approaches. We revealed that the aminoglycoside- mediated read-through of MC4R nonsense mutations induces a (party) restoration of the protein expression and receptor function in vitro, depending on several different factors. For further understanding of genetically determined obesity and for the development of new diagnostics and treatment, additional analyses, including polygenic effects, are necessary

Inverse agonistic action of 3-iodothyronamine at the human trace amine-associated receptor 5.

Application of 3-iodothyronamine (3-T1AM) results in decreased body temperature and body weight in rodents. The trace amine-associated receptor (TAAR) 1, a family A G protein-coupled receptor, is a target of 3-T1AM. However, 3-T1AM effects still persist in mTaar1 knockout mice, which suggest so far unknown further receptor targets that are of physiological relevance. TAAR5 is a highly conserved TAAR subtype among mammals and we here tested TAAR5 as a potential 3-T1AM target. First, we investigated mouse Taar5 (mTaar5) expression in several brain regions of the mouse in comparison to mTaar1. Secondly, to unravel the full spectrum of signaling capacities, we examined the distinct Gs-, Gi/o-, G12/13-, Gq/11- and MAP kinase-mediated signaling pathways of mouse and human TAAR5 under ligand-independent conditions and after application of 3-T1AM. We found overlapping localization of mTaar1 and mTaar5 in the amygdala and ventromedial hypothalamus of the mouse brain. Second, the murine and human TAAR5 (hTAAR5) display significant basal activity in the Gq/11 pathway but show differences in the basal activity in Gs and MAP kinase signaling. In contrast to mTaar5, 3-T1AM application at hTAAR5 resulted in significant reduction in basal IP3 formation and MAP kinase signaling. In conclusion, our data suggest that the human TAAR5 is a target for 3-T1AM, exhibiting inhibitory effects on IP3 formation and MAP kinase signaling pathways, but does not mediate Gs signaling effects as observed for TAAR1. This study also indicates differences between TAAR5 orthologs with respect to their signaling profile. In consequence, 3-T1AM-mediated effects may differ between rodents and humans

Investigation of Naturally Occurring Single-Nucleotide Variants in Human TAAR1

Activation of trace amine-associated receptor 1 (TAAR1) in endocrine pancreas is involved in weight regulation and glucose homeostasis. The purpose of this study was the identification and characterization of potential TAAR1 variants in patients with overweight/obesity and disturbed glucose homeostasis. Screening for TAAR1 variants was performed in 314 obese or overweight patients with impaired insulin secretion. The detected variants were functionally characterized concerning TAAR1 cell surface expression and signaling properties and their allele frequencies were determined in the population-based Study of Health in Pomerania (SHIP). Three heterozygous carriers of the single nucleotide missense variants p.Arg23Cys (R23C, rs8192618), p.Ser49Leu (S49L, rs140960896), and p.Ille171Leu (I171L, rs200795344) were detected in the patient cohort. While p.Ser49Leu and p.Ille171Leu were found in obese/overweight patients with slightly impaired glucose homeostasis, p.Arg23Cys was identified in a patient with a complete loss of insulin production. Functional in vitro characterization revealed a like wild-type function for I171L, partial loss of function for S49L and a complete loss of function for R23C. The frequency of the R23C variant in 2018 non-diabetic control individuals aged 60 years and older in the general population-based SHIP cohort was lower than in the analyzed patient sample. Both variants are rare in the general population indicating a recent origin in the general gene pool and/or the consequence of pronounced purifying selection, in line with the obvious detrimental effect of the mutations. In conclusion, our study provides hints for the existence of naturally occurring TAAR1 variants with potential relevance for weight regulation and glucose homeostasis

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 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

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

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

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

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