J DINTER, JMU¨ HLHAUS and others 3-T1AM-modulated signaling 54:3 205–216 Research at ADRA2A 3-iodothyronamine differentially modulates a-2A-adrenergic receptor-mediated signaling Juliane Dinter1,*, Jessica Mu¨hlhaus1,*, Simon Friedrich Jacobi1,2, Carolin Leonie Wienchol1, Maxi Co¨ster3, Jaroslawna Meister3, Carolin Stephanie Hoefig2, Anne Mu¨ller1, Josef Ko¨hrle4, Annette Gru¨ters1, Heiko Krude1, Jens Mittag2, Torsten Scho¨neberg3, Gunnar Kleinau1 and Heike Biebermann1 1Institut fu¨ r Experimentelle Pa¨ diatrische Endokrinologie, Charite´ -Universita¨ tsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany 2Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Correspondence 3Institut fu¨ r Biochemie, Molekulare Biochemie, Medizinische Fakulta¨ t, University of Leipzig, Leipzig, Germany should be addressed 4Institut fu¨ r Experimentelle Endokrinologie, Charite´ -Universita¨ tsmedizin Berlin, Augustenburger Platz 1, to H Biebermann 13353 Berlin, Germany Email *(J Dinter and J Mu¨ hlhaus contributed equally to this work) [email protected] Abstract Most in vivo effects of 3-iodothyronamine (3-T1AM) have been thus far thought to be Key Words mediated by binding at the trace amine-associated receptor 1 (TAAR1). Inconsistently, the " G protein-coupled receptor 3-T1AM-induced hypothermic effect still persists in Taar1 knockout mice, which suggests " adrenergic receptor additional receptor targets. In support of this general assumption, it has previously been " thyronamine reported that 3-T1AM also binds to the a-2A-adrenergic receptor (ADRA2A), which " 3-T1AM modulates insulin secretion. However, the mechanism of this effect remains unclear. Journal of Molecular Endocrinology 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 Gi/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 Gi/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 Gi/o pathway but an antagonistic effect on MAPK induced by NorEpi. Moreover, in ADRA2A/TAAR1 hetero-oligomers, the capacity of NorEpi to stimulate Gi/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 Journal of Molecular receptors. Endocrinology (2015) 54, 205–216 http://jme.endocrinology-journals.org Ñ 2015 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-15-0003 Printed in Great Britain Downloaded from Bioscientifica.com at 09/25/2021 05:28:40AM via free access Research J DINTER, JMU¨ HLHAUS and others 3-T1AM-modulated signaling 54:3 206 at ADRA2A Introduction 3-iodothyronamine (3-T1AM) is a decarboxylated and 2 h following 3-T1AM application, and, in turn, to reduce deiodinated derivative of the classical thyroid hormone insulin concentration (Regard et al. 2007). Therefore, we levothyroxine (Scanlan 2009), and it was originally additionally examined the long-term effect of 3-T1AM isolated from rat brain homogenates, but it is also application in vivo in order to examine whether insulin detectable in peripheral blood and various peripheral secretion remains blocked after 3-T1AM stimulation of organs, such as the liver and heart (Scanlan et al. 2004, Adra2a. Finally, our analysis revealed important Saba et al. 2010, Hoefig et al. 2011, Zucchi et al. 2014). functional aspects of 3-T1AM-mediated signaling by Application of 3-T1AM in rodents, for example, results in interaction with ADRA2A and provided detailed insights a decreased body temperature (Scanlan et al.2004), into a diverse and extended set of ligand–receptor interplays. mediates a switch between lipid and glucose metabolism for energy consumption (Braulke et al. 2008), and shows dose-dependent effects on feeding behavior and body Material and methods weight (Dhillo et al. 2009, Manni et al. 2012, Haviland et al. 2013). In vitro, the receptor target of 3-T1AM is assigned to Cloning of ADRA2A and TAAR1 the trace amine-associated receptor 1 (TAAR1). 3-T1AM All human full-length constructs were amplified from activates TAAR1 via the Gs/adenylyl cyclase system genomic DNA or purchased (ADRA2A, Missouri S&T cDNA (Scanlan et al.2004). Inconsistently, the previously Resource Center, Rolla, MO, USA) and cloned into the reported 3-T1AM effects on thermoregulation (Scanlan eukaryotic expression vector pcDps. Constructs were et al. 2004), which are hypothermic, still persist in mTaar1 N-terminally tagged with a hemagglutinin (50-YPYDVP- knockout mice (Panas et al. 2010), which suggests that DYA-30) epitope (NHA) or C-terminally tagged with a Flag there are additional receptor targets in vivo. It has been epitope (C-Flag, 50-DYKDDDDK-30). Plasmids were previously reported that 3-T1AM also binds to the a-2A- sequenced and verified by BigDye-terminator sequencing adrenergic receptor (ADRA2A), a receptor that influences (PerkinElmer, Inc., Waltham, MA, USA) using an auto- glucose homeostasis (Regard et al. 2007), but the under- matic sequencer (ABI 3710xl; Applied Biosystems). lying mechanism is still unclear. We consequently hypothesized that signaling of ADRA2A is induced or modulated in the presence of 3-T1AM. In the present Cell culture and transient transfection Journal of Molecular Endocrinology study, we assumed two possibilities for modulation: an For cAMP and serum response element-luciferase (SRE-luc) influence of 3-T1AM either on ADRA2A alone or on a measurements, HEK293 cells were seeded in poly-L-lysine- putative ADRA2A/TAAR1 hetero-oligomer. Interaction of coated (Biochrom AG, Berlin, Germany) 96-well plates ADRA2A with other G protein-coupled receptors (GPCRs) (15 000 cells/well). Transient transfection of HEK293 cells has been demonstrated to lead to uncoupling of the was performed 24 h after seeding in supplement-free ADRA2A signal transduction pathway (Vilardaga et al. advanced MEM (Life Technologies) using Metafectene 2008). To test both possibilities, we characterized the (Biontex, Munich, Germany) according to the manu- signal transduction of ADRA2A and ADRA2A/TAAR1 facturer’s protocol. hetero-oligomers in response to 3-T1AM respectively. We first used the label-free EPIC technology (measure- Determination of Adra2a and Taar1 expression in ment of dynamic mass redistribution) and detected direct pancreatic islets by RNA sequencing analysis or indirect signaling modulation by 3-T1AM at ADRA2A. Classically, ADRA2A signals via Gi/o (Bylund & Ray- To determine GPCR expression levels in pancreatic islets, Prenger 1989). Therefore, we expressed ADRA2A in data from a recently performed RNA sequencing analysis HEK293 cells and tested receptor signaling related to Gi/o were utilized (Meister et al. 2014). In brief, total RNA from and MAPKs directly and in combination with norepine- isolated pancreatic islets (ten WT male 129S6 mice) was phrine (NorEpi), the endogenous agonist of ADRA2A. extracted and cDNA libraries were generated using TruSeq Complementary to this, we characterized the capacity of RNA Sample Preparation Kits v2 (Illumina, San Diego, CA, TAAR1 and ADRA2A to form hetero-oligomers. Moreover, USA) according to the manufacturer’s protocol. Indexed 3-T1AM has been shown to demonstrate high affinities to islet libraries of good quality were pooled and used for pancreatic islets, to increase blood glucose concentration sequencing on two flow cell lanes on an Illumina http://jme.endocrinology-journals.org Ñ 2015 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-15-0003 Printed in Great Britain Downloaded from Bioscientifica.com at 09/25/2021 05:28:40AM via free access Research J DINTER, JMU¨ HLHAUS and others 3-T1AM-modulated signaling 54:3 207 at ADRA2A HiScanSQ System. Trimmed paired-end reads were Determination of hetero-oligomerization by mapped to the reference mouse genome (July 2007 sandwich-ELISA NCBI37/mm9) with Ensembl v66 annotations using Receptor–receptor oligomerization was determined by a Tophat 1.3.3. (Trapnell et al. 2012), which aligns reads sandwich-ELISA as described previously (Piechowski et al. using Bowtie (version 0.9.9). Fragments per kilobase of 2013). In brief, N-terminally HA-tagged ADRA2A and transcript per million mapped reads (FPKM) values were C-terminally Flag-tagged TAAR1 (or vice versa)were calculated using Cufflinks version 1.3.0 (Roberts et al. co-expressed in COS-7 cells. These cells were selected 2011, Trapnell et al. 2012). because they are more robust than HEK293 cells, which is beneficial in light of the numerous washing steps of the Measurement of cAMP accumulation ELISA. The growth hormone secretagogue receptor (GHSR) homo-oligomer served as a positive control Gs and Gi/o signaling were determined by measuring cAMP (Rediger et al. 2011), whereas N-HA-tagged/C-Flag-tagged accumulation. Forty-eight hours following transfection, rat muscarinic 3 receptor (rM3R)