Identification of potential adrenergic receptors and genes involved in monoamine synthesis

For identification of potential adrenergic receptors, we first used the human adrenergic receptor gene (ID number: P35348/P35368/P25100/ P08913/ P18089/ P18825/ P08588/ P07550/ P13945) to BLAST candidate genes within the basal metazoans. This included species such as Nematostella vectensis (GCF_932526225.1), Amphimedon queenslandica (GCF_000090795.2), Mnemiopsis leidyi (AGCP00000000), Trichoplax adhaerens (ABGP00000000), Euplotes vannus (EVDB, http://evan.ciliate.org), Chlamydomonas reinhardtii (GCF_000002595.2), Monosiga brevicollis (GCF_000002865.3), Salpingoeca_rosetta (GCF_000188695.1). Genes with BLAST alignment E-values less than 1e⁻⁵ were selected as candidates. We then further BLASTed these hits against the human genome to eliminate genes whose top five hits did not include adrenergic receptor genes. Additionally, we used human Frizzled family genes, another GPCR family, as the outgroup for phylogenetic analysis. The protein sequences were aligned using MAFFT⁵⁴. Mega 11 was used to construct the maximum likelihood and Bayesian phylogenetic trees. The Maximum Likelihood Tree’s robustness was tested with 1000 ultrafast bootstrap replicates. Trees were visualized and annotated in Adobe Illustrator. Similarly, human genes related to monoamine synthesis, including DBH (ID:NP_000778.3), DDC (ID:NP_000781.2), PAH (ID:NP_000268.1), and TH (ID:NP_000351.2), were used to blast candidate genes within basal metazoans. The identified genes were further blasted back to the human genome database to eliminate false orthologues. The Maximum Likelihood Tree’s robustness was tested with 1000 ultrafast bootstrap replicates. Finally, the maximum likelihood method was used to construct the phylogenetic tree.