PROJECT SUMMARY Substance use disorders (SUDs) drive persistent and long-lasting changes in the biochemical makeup of neurons in the brain. For example, chronic cocaine use is known to cause the up-regulation of specific gene networks, some of which are correlated across humans and animal SUD models. Prior in vivo studies in animal SUD models have focused on mapping RNA changes within specific cell-types. However, it is the proteins (and not the RNA) that serve as the final druggable molecules that directly alter neuronal function and can potentially treat SUDs. Thus, it is critical to develop novel methodologies that aim to identify molecular mechanisms of SUDs at the protein level within specific neuronal cell-types. Previous work has engineered proximity-labeling enzymes (e.g. BioID, TurboID) that can chemically label nearby proteins with a small biotin molecule. By expressing these enzymes within specific subcellular compartments within the neurons, it is possible to tag and then enrich biotinylated proteins that are present within that sub-compartment. However, a major limitation of these tools is that they are not activity-dependent; they will biotinylate proteins within a ~10- 20nm radius regardless of the cell-state or nearby receptor activation. Therefore, it is challenging to use them to identify proteins that are dynamically recruited to an activated GPCR in response to drug use in vivo. Here we will engineer new activity-dependent split-TurboID probes that will allow tagging of proteins nearby a drug- activated GPCR. These proteins can then be enriched and analyzed using liquid chromatography tandem mass-spectrometry (LC-MS/MS). First, we will use protein engineering, mutagenesis, and screening to design a new split-TurboID enzyme pair that becomes functional upon ligand binding to DRD1. Next, we will carry out in vivo proteomic studies using both 1) a full-length TurboID tethered to the C-terminus of DRD1 (independent from Aim 1, and 2) the DRD1 split-TurboID enzyme pair from Aim 1. As a proof of principle, we expect to identify distinct, acute DRD1 interactomes that are present after acute versus prolonged exposure to cocaine, and after withdrawal from prolonged exposure to cocaine. More broadly, this technology will enable the unbiased identification of intracellular proteins that are recruited to DRD1 in response to substances that modulate dopamine binding. These aims will establish new and transformative technologies for sensitive in vivo proteomic tagging, and in the future can be used to identify mechanisms underlying SUDs along the continuum of addiction.