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

Functional selectivity of G-Protein coupled receptors with a role in energy homeostasis

G-Protein gekoppelte Rezeptoren (GPCRs) sind an der Steuerung aller grundlegenden Zell- und Körperfunktionen wie Metabolismus, Zelldifferenzierung und Wachstum beteiligt. Die Eigenschaften und Mechanismen der GPCRs sind komplex, wobei jeder einzelne Rezeptor individuell zu betrachten und im Zusammenhang zu untersuchen ist. In dieser Arbeit wurden Rezeptoren erforscht, die insbesondere in der Energiehomöostase eine Rolle spielen und Lücken im Verständnis ihrer Funktionalität aufweisen. Der Fokus wurde auf fünf ausgewählte GPCRs gelegt, den G-Protein gekoppelten Rezeptor 83 (GPR 83), den Melanocortin-4 Rezeptor (MC4R), die beta-adrenergen Rezeptoren 1 und 2 (ADRB1 und ADRB2), sowie den Trace amine-assoziierten Rezeptor 1 (TAAR1). Es sollten Signalwege des orphanen (Ligand unbekannt) GPR83, Interaktions- Regulationsmechanismen des wichtigsten GPCR in der Appetitregulation - MC4R, sowie dfferentielle Signalisierungsmodulationen an ADRB1 und ADRB2 untersucht werden. Im Rahmen der genannten Projekte und der hier aufgeführten resultierenden Publikationen wurden die wichtigsten Signalwege funktionell mittels zellbasierter Stimulationsversuche charakterisiert. Zum anderen wurden GPCR-GPCR Interaktionen durch die Methode des Sandwich-ELISA überprüft. Die Untersuchungen zeigten, dass der GPR83 im Zusammenspiel mit Zink (II) den Gq/11-Signalweg aktiviert, basal aktiv (Liganden unabhängig) ist, sich für Gq/11 durch bestimmte Mutationen auch konstitutiv aktivieren lässt und als Homodimer vorliegt. Durch die Studien zum MC4R konnte festgestellt werden, dass die intrazelluläre Schleife 2 und Teile der verbundenen Transmembranhelizes 3 und 4 zur Bildung von MC4R-Homodimeren essentiell sind. Durch eine gezielte Separierung des Homodimers durch Modifikationen an diesen Komponenten wurde eine gesteigerte Liganden unabhängige Basalaktivität erzeugt, was auf einen inhibitorischen Einfluss durch die Dimerisierung schlieÿen lässt. Die Spurenamine Tyramin, beta-Phenylethylamin und Octopamin sind nicht nur Agonisten des TAAR1, sondern wirken auch als partielle Antagonisten oder Agonisten an ADRB1 und ADRB2. Die differentielle Modulation zeigte sich entweder in einer Blockade oder in einer Aktivierung des Gs- Signalweges. Strukturmodellbetrachtungen im Zusammenspiel mit den funktionellen Daten führten zu der Schlussfolgerung, dass die gemeinsamen Liganden von TAAR1, ADRB1 und ADRB2 aufgrund einer hohen Aminosäureähnlichkeit in einer bestimmten Ligandenbindungsregion lokalisiert sind und ortho- oder allosterisch wirken. Zusammenfassend führten alle drei Hauptprojekte zu neuen Erkenntnissen, auf die in weiteren Studien aufgebaut werden kann. Es wurden insbesondere neue Details zur funktionellen Selektivität von GPCRs in der komplexen Regulation gezeigt, die nicht zuletzt helfen können, in das Problem des zunehmenden Übergewichts weiter Teile der Weltbevölkerung zielgerichtet und selektiv pharmakologisch einzugreifen.G-protein coupled receptors (GPCRs) are involved in the control of all basic cellular and physiological functions such as metabolism, cell differentiation and growth. The characteristics and mechanisms of GPCRs are complex, while each individual receptor needs to be investigated individually and considered in context to each other. Within this paper receptors were ascertained which are particularly involved in the energy homeostasis but which still hold gaps in understanding their functionality. The focus was placed on five selected GPCRs, the G-protein coupled receptor 83 (GPR 83), the melanocortin-4 receptor (MC4R), the beta-adrenergic receptors 1 and 2 (ADRB1 and ADRB2), and the trace amine-associated receptor 1 (TAAR1). The aim was to unravel signaling pathways of the orphan (ligand unknown) GPR83, regulatory interaction mechanisms of the most important GPCR in appetite regulation of the MC4R, and differential signaling modulations of ADRB1 and ADRB2. In the scope of these projects and the resulting publications listed here the most important signaling pathways were functionally characterized using cell-based stimulation assays. On the other hand GPCR-GPCR interactions were investigated using a sandwich-ELISA approach. The investigations showed that the GPR83 is activated in combination with zinc (II) in the Gq/11-signaling pathway, is basally active (ligand independent), can be activated constitutively for Gq/11 by a certain mutation, and moreover is present as homodimer. Through the MC4R studies it was shown that the intracellular loop 2 and parts of the conjoined transmembrane helices 3 and 4 are essential for MC4R homodimer formation. Modifications of these components led to a selective separation of the homodimers and increased the ligand independent basal activity suggesting an inhibitory influence by the dimerization. The trace amines tyramine, beta-phenylethylamine and octopamine are not only agonists at TAAR1, but also act as partial antagonists or agonists at ADRB1 and ADRB2. The differential modulation was exhibited in either a blockade or an activation of the Gs-signaling pathway. Consideration of structure models in combination with the obtained functional data led to the conclusion that the shared ligands of TAAR1, ADRB1 and ADRB2 are localized in a specific ligand binding region due to their high amino acid similarity, thus acting ortho- or allosterically. In summary, all three main projects led to new insights providing information and knowledge for further studies. New details unraveling the functional selectivity of GPCRs in the complex regulation are shown which may help to reduce the increasing obesity as problem of the world population by intervening with selective pharmaceuticals

Die Verbesserung der touristischen Wettbewerbsfähigkeit von ländlichen Destinationen. GW-Unterricht|GW-Unterricht 144

In vielen ländlichen Regionen spielt der Tourismus eine wichtige Rolle. Diese Rolle (Arbeitsplätze, Einkommen etc.) ist gefährdet, weil immer mehr ländliche Destinationen Mühe bekunden, im globalisierten Tourismusmarkt konkurrenzfähig zu bleiben. Im vorliegenden Artikel wird die touristische Wettbewerbsfähigkeit von zwei ländlichen Destinationen in Niederösterreich mit Hilfe der Importance-Performance-Analyse untersucht. Ziel ist es, die Stärken und Schwächen der beiden Regionen in Bezug auf ihre Wettbewerbsfähigkeit herauszuarbeiten, aber auch die Vor- und Nachteile der angewandten Methodik zu diskutieren

Die Verbesserung der touristischen Wettbewerbsfähigkeit von ländlichen Destinationen. GW-Unterricht|GW-Unterricht 144

In vielen ländlichen Regionen spielt der Tourismus eine wichtige Rolle. Diese Rolle (Arbeitsplätze, Einkommen etc.) ist gefährdet, weil immer mehr ländliche Destinationen Mühe bekunden, im globalisierten Tourismusmarkt konkurrenzfähig zu bleiben. Im vorliegenden Artikel wird die touristische Wettbewerbsfähigkeit von zwei ländlichen Destinationen in Niederösterreich mit Hilfe der Importance-Performance-Analyse untersucht. Ziel ist es, die Stärken und Schwächen der beiden Regionen in Bezug auf ihre Wettbewerbsfähigkeit herauszuarbeiten, aber auch die Vor- und Nachteile der angewandten Methodik zu diskutieren

The Trace Amine-Associated Receptor 1 Agonist 3-Iodothyronamine Induces Biased Signaling at the Serotonin 1b Receptor

Trace amine-associated receptors (TAARs) belong to the class A G-protein-coupled receptors (GPCR) and are evolutionary related to aminergic receptors. TAARs have been identified to mediate effects of trace amines. TAAR1 signaling is mainly mediated via activation of the Gs/adenylyl cyclase pathway. In addition to classical trace amines, TAAR1 can also be activated by the thyroid hormone derivative 3-iodothyronamine (3-T1AM). Pharmacological doses of 3-T1AM induced metabolic and anapyrexic effects, which might be centrally mediated in the hypothalamus in rodents. However, the observed anapyrexic effect of 3-T1AM persists in Taar1 knock-out mice which raises the question whether further GPCRs are potential targets for 3-T1AM and mediate the observed physiological effect. Anapyrexia has been observed to be related to action on aminergic receptors such as the serotonin receptor 1b (5-HT1b). This receptor primarily activates the Gi/o mediated pathway and PLC signaling through the Gβγ of Gi/o. Since the expression profiles of TAAR1 and 5-HT1b overlap, we questioned whether 3-T1AM may activate 5-HT1b. Finally, we also evaluated heteromerization between these two GPCRs and tested signaling under co-expressed conditions. In this study, we showed, that 3-T1AM can induce Gi/o signaling through 5-HT1b in a concentration of 10 μM. Strikingly, at 5-HT1b the ligand 3-T1AM only activates the Gi/o mediated reduction of cAMP accumulation, but not PLC activation. Co-stimulation of 5-HT1b by both ligands did not lead to additive or synergistic signaling effects. In addition, we confirmed the capacity for heteromerization between TAAR1 and 5-HT1b. Under co-expression of TAAR1 and HTR1b, 3-T1AM action is only mediated via TAAR1 and activation of 5-HT1b is abrogated. In conclusion, we found evidence for 5-HT1b as a new receptor target for 3-T1AM, albeit with a different signaling effect than the endogenous ligand. Altogether, this indicates a complex interrelation of signaling effects between the investigated GPCRs and respective ligands

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

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