Adenylyl cyclases are key points for the concurrent integration of diverse signaling pathways. Controlling production of the second messenger cAMP, adenylyl cyclases provide an important mechanism for the regulation of physiological functions by amplifying signaling events to stimulate downstream effectors. While different isoforms of adenylyl cyclase exhibit distinct patterns of expression and regulation, of particular interest are two groups of Ca2+ regulated isoforms that are highly expressed in the central nervous system. Adenylyl cyclase type 5 (AC5) is a Ca2+ inhibited isoform that is highly expressed in the striatum, and whose activity is involved in the regulation of movement, pain, and metabolism. Adenylyl cyclase type 8 (AC8) is stimulated by Ca2+ in a calmodulin dependent manner, and appears to be involved with long-term memory, anxiety, and reward pathways. Studying the signaling characteristics of these adenylyl cyclase isoforms is necessary for improving our scientific understanding of biological pathways, as well identifying therapeutic targets that can be exploited for treatment of disease. In this work, we investigated changes in the protein interaction network of AC5 following prolonged Gi/o-mediated inhibition that results in heterologous sensitization. The diversity of signaling pathways and multitude of protein interactions that have been implicated in the development of the heterologous sensitization response prompted the development of a novel screening strategy to capture and identify AC5-protein interactions which occur following prolonged Gi/o-mediated inhibition. We utilized bimolecular fluorescence complementation (BiFC) in conjunction with fluorescence activated cell sorting (FACS) and Next Generation sequencing to capture, identify, and characterize novel AC5 interacting partners. We further studied the effects of increased AC5 activity by functionally characterizing a series of gain-of-function mutations that have been identified in patients diagnosed with Familial Dyskinesia and Facial Myokymia (FDFM). Our results demonstrate that the AC5 mutants exhibit enhanced activity to Gs-mediated stimulation and reduced inhibition by Gi/o-coupled receptors. We further suggest that this dysregulation of AC5 in striatal medium spiny neurons likely results in an imbalance in the direct and indirect striatal signaling pathways that coordinate the initiation and maintenance of movement. Genetic models of AC8 regulation have implicated its activity in signaling pathways that may regulate comorbid long-term anxiety and ethanol consumption. Therefore, we developed and conducted a high-throughput screen and validation paradigm of small molecules for the discovery of AC8 selective inhibitors. The screening effort identified two lead compounds that demonstrate enhanced efficacy and selectivity over AC1 compared to currently available adenylyl cyclase inhibitors.

The D3 dopamine receptor (D3R) belongs to D2-class of dopamine receptors (DARs) and is involved in emotion, movement, and reward. D3R dysfunction has been reported in some neuropsychiatric disorders such as addiction, cognitive deficits, depression, schizophrenia, and Parkinson’s disease. Genetic studies have shown two polymorphic variants of the D3R gene resulting from substitution of serine to glycine at position nine of the amino terminus. Isoform 5 of adenylyl cyclase (AC5) is one of the nine transmembrane bound ACs in the brain and myocardium. Previous studies in rats have shown that AC5 is expressed in the striatum, nucleus accumbens and olfactory tubercle and at lower levels in islands of Calleja, where the D3R is also expressed. Previous studies showed that although D2R and D4R inhibit ACs activity in different cell types, inhibition of ACs by D3R is weak and often undetectable. It has been shown that D3R selectively inhibits AC5 activity in human embryonic kidney 293 (HEK293) cells co-transfected with D3R and AC5. Co-expression of D3R and AC5 in brain regions which are major coordinators of normal and pathological movement, and the selective inhibition of AC5 activity by D3R raise the possibility of a functional link between AC5 and D3R in the modulation of signal transduction and trafficking. I hypothesized that AC5 plays a unique role in modulation of D3R trafficking and signaling pathways through interaction between D3R and AC5. Herein, I demonstrated an interaction between D3R and AC5 in vivo and in vitro using reciprocal co-immunoprecipitation/immunoblotting (co-IP/IB) assays. Interestingly, DA may facilitate the formation of protein complex between D3R and AC5 in vitro. Radio ligand binding assays revealed that heterodimerization of D3R polymorphic variants with AC5 does not change ligand binding affinity and expression of the D3R. Furthermore, taking advantages of GloSensor assays, selective inhibition of AC5 activity by D3Ser9 and D3Gly9 has been shown following activation by DA and quinpirole. Using ELISA studies showed that AC5 promotes cell surface expression and total expression of D3Ser9 and D3Gly9. Moreover, ELISA results suggested that AC5 facilitates DA-induced D3Ser9 endocytosis in dynamin and β-arrestin 2 dependent process, while having no effect on D3Gly9 polymorphic variant. The results also revealed that AC5 attenuates heterologous (PKC-induced) internalization of D3Ser9, while it does not have any effect on D3Gly9 heterologous internalization. My results also displayed a complex formation between D3R, AC5 and, β-arrestin 2 under basal and DA stimulation conditions, which emphasize the role of β-arrestin 2 in D3R signal transduction. Overall, a new regulatory mechanism for D3R has been suggested. My results suggested that complex formation between both D3R polymorphic variants with AC5 can regulate signaling and trafficking properties of D3R without changing the binding affinity of the receptor. These data will be meaningful for understanding of diseases and developing treatment strategies.