Supplementary Material for: Evolutionary Conservation of 3-Iodothyronamine as an Agonist at the Trace Amine-Associated Receptor 1

<b><i>Objectives:</i></b> The trace amine-associated receptor 1 (Taar1) is a G<sub>s</sub> protein-coupled receptor activated by trace amines, such as β-phenylethylamine (β-PEA) and 3-iodothyronamine (T<sub>1</sub>AM). T<sub>1</sub>AM is an endogenous biogenic amine and thyroid hormone derivative that exerts several biological functions. However, the physiological relevance of T<sub>1</sub>AM acting via Taar1 is still under discussion. Therefore, we studied the structural and functional evolution of Taar1 in vertebrates to provide evidence for a conserved Taar1-mediated T<sub>1</sub>AM function. <b><i>Study Design:</i></b> We searched public sequence databases to retrieve Taar1 sequence information from vertebrates. We cloned and functionally characterized Taar1 from selected vertebrate species using cAMP assays to determine the evolutionary conservation of T<sub>1</sub>AM action at Taar1. <b><i>Results:</i></b> We found intact open reading frames of Taar1 in more than 100 vertebrate species, including mammals, sauropsids and amphibians. Evolutionary conservation analyses of Taar1 protein sequences revealed a high variation in amino acid residues proposed to be involved in agonist binding, especially in rodent Taar1 orthologs. Functional characterization showed that T<sub>1</sub>AM, β-PEA and <i>p-</i>tyramine (<i>p-</i>Tyr) act as agonists at all tested orthologs, but EC<sub>50</sub> values of T<sub>1</sub>AM at rat Taar1 differed significantly when compared to all other tested vertebrate Taar1. <b><i>Conclusions:</i></b> The high structural conservation of Taar1 throughout vertebrate evolution highlights the physiological relevance of Taar1, but species-specific differences in T<sub>1</sub>AM potency at Taar1 orthologs suggest a specialization of rat Taar1 for T<sub>1</sub>AM recognition. In contrast, β-PEA and <i>p-</i>Tyr potencies were rather conserved throughout all tested Taar1 orthologs. We provide evidence that the observed differences in potency are related to differences in constraint during Taar1 evolution.