PROJECT SUMMARY Dysregulation of dopamine (DA) signaling is thought to underlie the neural deficits of serious mental illness (SMI), including schizophrenia, bipolar disorder, and depression. Treatment for SMI relies on second generation antipsychotics (SGAs), which bind with high affinity to the DA D2-like family of inhibitory Gi/o trimeric G protein coupled receptors, thereby impairing the ability of DA receptors to signal to G protein in response to DA. While the primary target of SGAs was thought to be the D2 DA receptor (D2R), in-vitro binding assays have demonstrated equal or even higher affinity of these drugs for the D3 DA receptor (D3R), which is known to be localized in neural loci associated with affect. Recently, we found that D3Rs in the axon initial segment (AIS) of midbrain and prefrontal cortex modulate cellular excitability via an Arrestin-mediated, ERK1/2 dependent interaction with T-type Ca2+ channels (CaV3.2), as opposed to canonical Gi-mediated Ca2+ channel inhibition. This is particularly intriguing as DA binding to D3R does not promote Arrestin recruitment except, as we have found, with concomitant activation of protein kinase C (PKC) via second messengers and/or depolarization. Furthermore, we have found that some SGAs engage this Arrestin-specific signaling mechanism even in the absence of G protein activation. I hypothesize that ligand binding and PKC activation promote independent phosphorylation events on the D3R, both of which are required for Arrestin engagement and channel modulation. Here, I will examine the molecular determinants of the neuromodulatory interaction between the D3R and CaV3.2. I will determine the ligand-dependent and PKC-dependent kinase phosphorylation sites on the D3R that are integral to this functional interaction using site-directed mutagenesis. I will create heterologous cell lines stably expressing these mutant receptors and examine their impact on D3R Ca2+ modulation using in vitro whole cell patch clamp electrophysiology in this heterologous system. I will also examine the ability of a panel of SGAs to recruit Arrestin and modulate channel function. Lastly, I will examine which SGA ligands, as a consequence of Arrestin engagement, promote D3R endocytosis and degradation upon prolonged drug administration, and whether this differential trafficking can account for the variable cognitive side-effects commonly observed in patient populations, using in vivo mouse behavioral models. I hypothesize that while all SGAs antagonize G protein signaling, only some will engage Arrestin supporting acute inhibition of CaV3.2 in the AIS while others will not. By extension, I also hypothesize that the select ligands that engage Arrestin will promote D3R downregulation during repeated SGA administration while those that do not will promote D3R upregulation by preventing DA-mediated endocytosis and degradation. My goal is to better characterize SGAs for their D3R Arrestin-mediated signaling and trafficking, a...