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Analysis of human TAAR8 and murine Taar8b mediated signaling pathways and expression profile.

Mühlhaus J, Dinter J, Nürnberg D, Rehders M, Depke M, Golchert J, Homuth G, Yi CX, Morin S, Köhrle J, Brix K, Tschöp M, Kleinau G, Biebermann H - Int J Mol Sci (2014)

Bottom Line: 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 G(s) signaling in vitro.Interestingly, 3-T1AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T1AM might exist.We could not identify 3-T1AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal G(i/o) signaling activity, a so far unknown signaling pathway for TAARs.

View Article: PubMed Central - PubMed

Affiliation: Institut für Experimentelle Pädiatrische Endokrinologie, Charité-Universitätsmedizin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany. jessica.muehlhaus@charite.de.

ABSTRACT
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 G(s) 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 G(s) and/or G(i/o) signaling. Activation of G-proteins G(q/11) and G(12/13) 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 G(i/o) signaling activity, a so far unknown signaling pathway for TAARs.

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Expression studies of Taar8b in murine heart using in situ hybridization. Expression studies were performed by in situ hybridization (ISH) using a LNA (locked nucleic acid) probe. C57BL/6 WT mouse hearts were sectioned and treated with the LNA probe. Signals were visualized with an avidin–biotin complex using DAB (3,3'-diaminobenzidin) (A–C) or DY-light 488 streptavidin (D) staining. (A) The mGata probe used as a positive control caused significant staining of the nucleus and the perinuclear region of cardiomyocytes, as expected; (B) The negative control performed by hybridization with scrambled ISH probe demonstrated homogenous shading; (C) Treatment with Taar8b specific probes did not result in staining of the heart tissue, pointing to a lack of expression of Taar8b in the murine heart; (D) COS-7 cells transiently transfected with Taar8b served as a positive control for the Taar8b probe, exhibiting prominent DyLight-488 positive signal , thereby proving functionality of the probe.
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ijms-15-20638-f002: Expression studies of Taar8b in murine heart using in situ hybridization. Expression studies were performed by in situ hybridization (ISH) using a LNA (locked nucleic acid) probe. C57BL/6 WT mouse hearts were sectioned and treated with the LNA probe. Signals were visualized with an avidin–biotin complex using DAB (3,3'-diaminobenzidin) (A–C) or DY-light 488 streptavidin (D) staining. (A) The mGata probe used as a positive control caused significant staining of the nucleus and the perinuclear region of cardiomyocytes, as expected; (B) The negative control performed by hybridization with scrambled ISH probe demonstrated homogenous shading; (C) Treatment with Taar8b specific probes did not result in staining of the heart tissue, pointing to a lack of expression of Taar8b in the murine heart; (D) COS-7 cells transiently transfected with Taar8b served as a positive control for the Taar8b probe, exhibiting prominent DyLight-488 positive signal , thereby proving functionality of the probe.

Mentions: As it is known that TAARs, in general, are expressed at low levels except for in the olfactory epithelium [7,26], we applied the very sensitive method “locked nucleic acid” (LNA) in situ hybridization (ISH) for the example of heart tissue. In this particular case, we were able to demonstrate the absence of Taar8b mRNA for this tissue (Figure 2).


Analysis of human TAAR8 and murine Taar8b mediated signaling pathways and expression profile.

Mühlhaus J, Dinter J, Nürnberg D, Rehders M, Depke M, Golchert J, Homuth G, Yi CX, Morin S, Köhrle J, Brix K, Tschöp M, Kleinau G, Biebermann H - Int J Mol Sci (2014)

Expression studies of Taar8b in murine heart using in situ hybridization. Expression studies were performed by in situ hybridization (ISH) using a LNA (locked nucleic acid) probe. C57BL/6 WT mouse hearts were sectioned and treated with the LNA probe. Signals were visualized with an avidin–biotin complex using DAB (3,3'-diaminobenzidin) (A–C) or DY-light 488 streptavidin (D) staining. (A) The mGata probe used as a positive control caused significant staining of the nucleus and the perinuclear region of cardiomyocytes, as expected; (B) The negative control performed by hybridization with scrambled ISH probe demonstrated homogenous shading; (C) Treatment with Taar8b specific probes did not result in staining of the heart tissue, pointing to a lack of expression of Taar8b in the murine heart; (D) COS-7 cells transiently transfected with Taar8b served as a positive control for the Taar8b probe, exhibiting prominent DyLight-488 positive signal , thereby proving functionality of the probe.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264187&req=5

ijms-15-20638-f002: Expression studies of Taar8b in murine heart using in situ hybridization. Expression studies were performed by in situ hybridization (ISH) using a LNA (locked nucleic acid) probe. C57BL/6 WT mouse hearts were sectioned and treated with the LNA probe. Signals were visualized with an avidin–biotin complex using DAB (3,3'-diaminobenzidin) (A–C) or DY-light 488 streptavidin (D) staining. (A) The mGata probe used as a positive control caused significant staining of the nucleus and the perinuclear region of cardiomyocytes, as expected; (B) The negative control performed by hybridization with scrambled ISH probe demonstrated homogenous shading; (C) Treatment with Taar8b specific probes did not result in staining of the heart tissue, pointing to a lack of expression of Taar8b in the murine heart; (D) COS-7 cells transiently transfected with Taar8b served as a positive control for the Taar8b probe, exhibiting prominent DyLight-488 positive signal , thereby proving functionality of the probe.
Mentions: As it is known that TAARs, in general, are expressed at low levels except for in the olfactory epithelium [7,26], we applied the very sensitive method “locked nucleic acid” (LNA) in situ hybridization (ISH) for the example of heart tissue. In this particular case, we were able to demonstrate the absence of Taar8b mRNA for this tissue (Figure 2).

Bottom Line: 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 G(s) signaling in vitro.Interestingly, 3-T1AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T1AM might exist.We could not identify 3-T1AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal G(i/o) signaling activity, a so far unknown signaling pathway for TAARs.

View Article: PubMed Central - PubMed

Affiliation: Institut für Experimentelle Pädiatrische Endokrinologie, Charité-Universitätsmedizin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany. jessica.muehlhaus@charite.de.

ABSTRACT
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 G(s) 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 G(s) and/or G(i/o) signaling. Activation of G-proteins G(q/11) and G(12/13) 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 G(i/o) signaling activity, a so far unknown signaling pathway for TAARs.

Show MeSH
Related in: MedlinePlus