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.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/43577 |
| Date | 10 May 2022 |
| Creators | Habibi Khorasani, Hedieh |
| Contributors | Tiberi, Mario |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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