PROJECT SUMMARY Opiate use, dependence and addiction represent enduring public health issues, resulting in substantial financial and societal health burdens, as well as increasing numbers of overdoses. Drug addiction is characterized as a chronic, relapsing disease. However, to date, there remains insufficient data examining the molecular mechanisms underlying persistent opiate-induced neurobiological changes, which has led to a scarcity of effective therapies and interventions to treat and prevent relapse. Drug addiction has long been thought of as a disorder of dopamine (DA) signaling. However, therapeutic interventions targeting receptor mediated DA neurotransmission have not yet resulted in fully efficacious treatments. Therefore, an overarching goal of our laboratories – and the focus of this application – has been to investigate novel, non-canonical actions of DA involved in mediating addiction phenotypes. Our laboratory recently identified a novel signaling moiety for DA in brain, termed dopaminylation (dop), whereby DA acts as a donor source for the establishment of post-translational modifications (PTM) on substrate proteins (e.g., histone H3) via transamidation by the Transglutaminase 2 (TGM2) enzyme. In more recent efforts by our lab to unbiasedly identify additional substrates of these PTMs (focused now on synaptic proteins in nucleus accumbens/NAc, a key brain reward region), we developed a novel chemical tagging approach that, when coupled to mass spectrometry, allowed for the discovery of hundreds of dopaminylated proteins in brain, both in the context of normal neural function and in response to aberrant dopamine signaling following chronic heroin self-administration (SA) in rats. Among them, gCaMKII: 1) was found to be robustly dopaminylated at only a single amino acid residue located within its autoinhibitory helix [glutamine (Q)285], a site that exists only two amino acids away from a critical threonine (T) residue (287), which when phosphorylated directs Calmodulin (CaM) sequestration; 2) is upregulated in its dopaminylation following heroin SA, both during acute and prolonged abstinence, but not in response to natural rewards; and 3) represents a critical substrate involved in mediating long range signals from the synapse to nucleus in brain, ultimately promoting CREB activation and neuronal plasticity. Thus, this dopaminylation event on gCaMKII may represent a critical convergent mechanism linking altered dopaminergic signaling in response to heroin to CREB mediated transcriptional abnormalities. As such, we hypothesize that gCaMKIIQ285dop may play a direct role in mediating heroin relapse via aberrant modulation of CREB signaling in NAc. In Aim 1, we will fully characterize gCaMKIIQ285dop’s temporal effects on drug taking vs. relapse vulnerability in the context of heroin SA. In Aim 2, we will explore gCaMKIIQ285dop’s effects on CREB signaling/transcription following heroin SA, events that may precipitate relapse vulnerabi...