Functional Deorphanization and Subtype-Selective Pharmacology of Three Tyramine Receptors in the Disease Vector, Aedes aegypti

Biogenic amines such as tyramine (TA) and octopamine (OA) are central regulators of insect physiology and behaviour, acting through G protein-coupled receptors (GPCRs) to control reproduction, locomotion, metabolism, olfaction and hydromineral homeostasis. Although TA was once considered solely a biosynthetic precursor to OA, it is now recognized as an independent signaling molecule acting through distinct tyramine receptors (TARs). Owing to their invertebrate-specific roles and absence in vertebrates, TARs represent promising molecular targets for selective insecticide development. In the mosquito Aedes aegypti, a major arboviral vector of dengue and Zika viruses, the functional and pharmacological properties of TARs have not been characterized. Here, we functionally deorphanized and comparatively characterized three putative A. aegypti tyramine receptors (AaTAR1-AaTAR3) using a heterologous assay, revealing subtype-specific pharmacological profiles and antagonist sensitivities. All three receptors were robustly activated by TA in a concentration-dependent manner, whereas OA exhibited significantly lower potency on each receptor subtype, consistent with a strong preference for TA. All three TARs were unresponsive to dopamine and serotonin, even when using supraphysiological concentrations, indicating high ligand specificity. Antagonist profiling revealed pronounced subtype-specific pharmacology: yohimbine strongly suppressed AaTAR1, phentolamine most effectively inhibited AaTAR2, whereas AaTAR3 exhibited reduced sensitivity to several classical aminergic antagonists, suggesting a pharmacologically distinct subtype. These findings establish that AaTAR1, AaTAR2 and AaTAR3 are bona fide functional tyramine receptors, define their ligand selectivity and subtype-specific pharmacology, and provide a comparative framework for understanding mosquito tyraminergic signaling, highlighting their potential as targets for next-generation vector control strategies.