Proximity Labeling of D1-Like Dopamine Receptors Captures Distinct Cellular Environments and Uncovers Trafficking Proteins That Regulate DA-Mediated Behaviors in Drosophila

Abstract The neurotransmitter dopamine (DA) is central to synaptic regulation that supports diverse behavioral functions, including both learning and forgetting. This multifunctional role of DA is due to receptor-specific signaling in specific subcellular environments that remain uncharacterized. Here we used Turbo-mediated proximity labeling in human cells to characterize the proximal environments of two Drosophila D1-like DA receptors (Dop1R1 and Dop1R2) in basal and DA-activation environments. DA drives both Dop1R1-Turbo and Dop1R2-Turbo to recruit β-arrestin 2, and Dop1R1-Turbo showed ligand-driven proximity to G-protein receptor kinase 3, members of clathrin-mediated endocytosis pathways, and WASH complex-mediated endosomal trafficking pathways. Additionally, we show evidence that Dop1R1 and Dop1R2 reside in distinct domains on the cell surface. In vivo disruption of Drosophila orthologues of Dop1R proximal proteins revealed three trafficking proteins, Sec24AB, Krz (beta-arrestin 2), and CG13887, that regulate R1-mediated learning, starvation-induced attraction to odors, and DA-mediated cAMP responses in memory circuits. In addition to revealing DA receptor trafficking proteins that support learning, our comparative characterization of the cellular environments of D1-like receptors offers insights into how DA differentially regulates diverse behavioral and synaptic functions.