Objective 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.

Most in vivo effects of 3-iodothyronamine (3-T1AM) have been thus far thought to be mediated by binding at the trace amine-associated receptor 1 (TAAR1). Inconsistently, the 3-T1AM-induced hypothermic effect still persists in Taar1 knockout mice, which suggests additional receptor targets. In support of this general assumption, it has previously been reported that 3-T1AM also binds to the a-2A-adrenergic receptor (ADRA2A), which modulates insulin secretion. However, the mechanism of this effect remains unclear. We tested two different scenarios that may explain the effect: the sole action of 3-T1AM at ADRA2A and a combined action of 3-T1AM at ADRA2A and TAAR1, which is also expressed in pancreatic islets. We first investigated a potential general signaling modification using the label-free EPIC technology and then specified changes in signaling by cAMP inhibition and MAPKs (ERK1/2) determination. We found that 3-T1AM induced G i/o activation at ADRA2A and reduced the norepinephrine (NorEpi)-induced MAPK activation. Interestingly, in ADRA2A/TAAR1 hetero-oligomers, application of NorEpi resulted in uncoupling of the G i/o signaling pathway, but it did not affect MAPK activation. However, 3-T1AM application in mice over a period of 6 days at a daily dose of 5 mg/kg had no significant effects on glucose homeostasis. In summary, we report an agonistic effect of 3-T1AM on the ADRA2A-mediated G i/o pathway but an antagonistic effect on MAPK induced by NorEpi. Moreover, in ADRA2A/TAAR1 hetero-oligomers, the capacity of NorEpi to stimulate G i/o signaling is reduced by co-stimulation with 3-T1AM. The present study therefore points to a complex spectrum of signaling modification mediated by 3-T1AM at different G protein-coupled receptors.Key Words " G protein-coupled receptor " adrenergic receptor

Background: 3-Iodothyronamine (3-T₁AM), a signaling molecule with structural similarities to thyroid hormones, induces numerous physiological responses including reversible body temperature decline. One target of 3-T₁AM is the trace amine-associated receptor 1 (TAAR1), which is a member of the rhodopsin-like family of G protein-coupled receptors (GPCRs). Interestingly, the effects of 3-T₁AM remain detectable in TAAR1 knockout mice, suggesting further targets for 3-T₁AM such as adrenergic receptors. Therefore, we evaluated whether β-adrenergic receptor 1 (ADRB1) and 2 (ADRB2) signaling is affected by 3-T₁AM in HEK293 cells and in human conjunctival epithelial cells (IOBA-NHC), where these receptors are highly expressed endogenously. Methods: A label-free EPIC system for prescreening the 3-T₁AM-induced effects on ADRB1 and ADRB2 in transfected HEK293 cells was used. In addition, ADRB1 and ADRB2 activation was analyzed using a cyclic AMP assay and a MAPK reporter gene assay. Finally, fluorescence Ca²⁺ imaging was utilized to delineate 3-T₁AM-induced Ca²⁺ signaling. Results: 3-T₁AM (10^-5- 10^-10M) enhanced isoprenaline-induced ADRB2-mediated G_s signaling but not that of ADRB1-mediated signaling. MAPK signaling remained unaffected for both receptors. In IOBA-NHC cells, norepinephrine-induced Ca²⁺ influxes were blocked by the nonselective ADRB blocker timolol (10 µM), indicating that ADRBs are most likely linked with Ca²⁺ channels. Notably, timolol was also found to block 3-T₁AM (10^-5M)-induced Ca²⁺ influx. Conclusions: The presented data support that 3-T₁AM directly modulates β-adrenergic receptor signaling. The relationship between 3-T₁AM and β-adrenergic signaling also reveals a potential therapeutic value for suppressing Ca²⁺ channel-mediated inflammation processes, occurring in eye diseases such as conjunctivitis.