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Dopamine based signaling makes up a small percentage of overall neuronal communication, but its regulation makes up a significant portion of CNS pharmacology. Dopamine is known to activate G protein-coupled receptors, which have been a drug target since at least the first modern antipsychotic (Chlorpromazine), although this was not clear at the time. Dopamine primarily binds a family of G protein-coupled receptors called the dopamine receptors. Dopamine receptors are either D1 or D2 -like, the significant difference being whether they bind and activate cyclic AMP production activating (D1-like) or inhibiting (D2-like) alpha subunits of G protein-coupled receptors. The D2-like receptors are the main targets of most antipsychotic and many anti-parkinsonian medicines currently in clinical use. 

We have investigated the downstream signaling of D2-like dopamine receptors D3 and D4. Specifically, we have used a nanoluciferase assay to measure G protein-coupled receptor interactions with intracellular proteins, primarily beta-arrestin2 (βarr2), G protein-coupled receptor kinase 2 (GRK2) and alpha subunits of G protein-coupled receptors. Included papers report on several intricacies of signaling downstream the D3 and D4 receptors. D4 receptor recruitment of GRK2 is dopamine dependent, transient and potentiates βarr2 recruitment. In addition, βarr2 desensitizes the response of G protein-coupled inward rectifier potassium (GIRK, also known as Kir3) channels to dopamine at the D4, this desensitization is similarly potentiated by GRK2 coexpression. This contrasts with the D3 receptor, where no dopamine-dependent GRK2 interaction could be detected. In time-resolved experiments, we found that the D3-selective agonist FAUC73 disassociates significantly faster from the D3 receptor than dopamine. We also investigated the common S9/G9 isoforms of the D3 receptor, with no significant difference found in either G protein, βarr2, or GRK2 recruitment, nor in downstream cAMP accumulation.

Additionally, we attempted to find D3 receptor mutants which only interact with Gαo subunits while not interacting with βarr2, and vice versa. The ultimate goal was to create mice with the corresponding D3 mutations, to allow for in vivo investigation of the behavioral consequences of the respective signaling pathways downstream of the D3 receptor. In this we have been partially successful with the discovery of the A131W point mutation, which seemingly renders the D3 receptor unable to signal though inhibitory G proteins while retaining βarr2 activity.

In preliminary experiments, c57BL/6 mice carrying the A131W point mutation have been tested using open field and prepulse inhibition paradigms. The goal of testing has been to compare the effects with wildtype and D3 knockout animals to pinpoint the pathways though which therapeutic drugs known to act via the D3 receptor, such as cariprazine, function downstream of the D3 receptor and to validate the efficacy of our model.

In conclusion, we set out to increase our understanding of how signaling downstream D3 and D4 receptors function. Beyond the basic science interest, we believe this knowledge might contribute to the development of novel therapies, as well as improving existing treatments acting via dopamine D2-like receptors, such as antipsychotics and antiparkinsonian drugs. Current receptor-level knowledge has allowed us to narrow the search for therapeutic targets, but continued progress will require research into the downstream signaling cascades. To facilitate this investigation, new tools need to be developed. Here, have adapted a nanoluciferase assay for use with D3 and D4 receptors to investigate protein-protein interactions between the receptors and GKR2, βarr2, and G protein Gα subunits (Gαo in particular). Using this method, we have identified a D3 receptor point mutation which disables G protein Gαo signaling while retaining βarr2 activity in vitro. Finally, we have created mutant c57BL/6 mice carrying this mutation and begun behavioral testing for comparison with wildtype (WT) and D3 knockout mice.