Proximity labelling of D1-like dopamine receptors reveals distinct cellular environments and uncovers trafficking proteins that regulate DA mediated behaviors in Drosophila

The neurotransmitter dopamine (DA) is central to synaptic regulation that support diverse behavioral functions, including both learning and forgetting. This multi-functional role of DA is due to receptor specific signaling in specific subcellular environments that remain uncharacterized. Here we utilized proximity labelling proteomics in human cells to characterize the proximal environments of two Drosophila D1-like DA receptors (Dop1R1 and Dop1R2) in basal and DA activation environments. While DA drives both receptors to recruit Beta-Arrestin 2, Dop1R1 alone showed ligand driven recruitment of G-protein Receptor Kinase 2/3, proximity to clathrin mediated endocytosis, and WASH complex mediated endosomal trafficking. Additionally, we show evidence that Dop1R1 and Dop1R2 reside in distinct domains at the cell surface. In vivo disruption of Drosophila orthologs of Dop1R proximal proteins revealed three trafficking proteins, Sec24AB, Krz, 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 D1-like receptors offers insights into how DA differentially regulates diverse behavioral and synaptic functions.