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Rol del receptor 3 de dopamina en la respuesta citotóxica generada en linfocitos T CD8+Chovar Vera, Ornella January 2018 (has links)
Seminario de Título para optar al Grado de:
Magíster en Ciencias Biológicas / El sistema nervioso y el sistema inmune se encuentran íntimamente relacionados
comunicándose bidireccionalmente mediante citoquinas, neuropéptidos y
neurotransmisores, como la dopamina. Este neurotransmisor ejerce su efecto a través de
cinco receptores dopaminérgicos que poseen diferentes grados de afinidad, contando con
la mayor afinidad el receptor 3 (D3R). Su presencia se ha reportado en diferentes células
del sistema inmune. La estimulación del D3R promueve la producción de IFN-γ en
linfocitos T CD4+, favoreciendo su diferenciación a un perfil Th1. Sin embargo, el rol de
este receptor en los linfocitos T CD8+ aún no se ha estudiado a cabalidad.
En esta tesis se estudió la incidencia del D3R en la formación del perfil citotóxico en
linfocitos T CD8+, que poseen un TCR transgénico específico para el péptido OVA(257-264)
alojado sobre el MHC de clase I H2-Kb (linfocitos OT-I). La diferenciación in vitro muestra
que la carencia del D3R en linfocitos OT-I (OT-I D3RKO) resulta en una reducción
significativa en la producción de IFN-γ, TNF-α, IL-2 y en la expresión de CD25 (cadena α
del receptor de IL-2) en respuesta a la estimulación con péptido OVA(257-264). Estas
deficiencias se revierten al añadir IL-2 exógena a los cultivos, sugiriendo que el
mecanismo de acción por el cual el D3R favorece la diferenciación hacia un fenotipo
efector productor de IFN-γ y TNF- α ocurre a través de la producción de IL-2. Concordante
con lo anterior, se observó que la expresión del D3R en linfocitos OT-I promueve la
expansión clonal y la protección antitumoral frente a melanoma.
En conjunto los resultados obtenidos en linfocitos T CD8+ sugieren que el D3R
promueve la formación de un perfil citotóxico, induciendo la producción de IFN-γ y
favoreciendo una alta tasa de expansión clonal, lo que conlleva a una mayor sobrevida
frente a un desafío tumoral. / The nervous system and the immune system are intimately related, communicating
bi-directionally through cytokines, neuropeptides and neurotransmitters. Dopamine is a
neurotransmitter that exerts its effect through five dopaminergic receptors which display
different degrees of affinity. The D3R, which display the highest affinity for dopamine, has
been found expressed in CD4+ T cells, where its stimulation promotes high production of
IFN-γ, favoring the acquisition of the Th1 profile. However, the role of this receptor in CD8+
T lymphocytes has not yet been fully studied.
In this thesis the role of D3R in the acquisition of the cytotoxic profile by CD8+ T
lymphocytes was studied. For this purpose, in this thesis CD8+ T-cells bearing a
transgenic TCR specific for the recognition of the OVA peptide(257-264) over the class I MHC
H2-Kb (OT-I lymphocytes) were used. In vitro differentiation experiments show that lack of
D3R in OT-I lymphocytes (OT-I D3RKO) results in a significant reduction in the production
of IFN-γ, TNF-α and on the expression of CD25 (α chain of the IL-2 receptor) in response
to the stimulation with OVA peptide(257-264). These effects were reversed by adding
exogenous IL-2 into the cultures, suggesting a mechanism by which D3R favors the
differentiation towards an effector phenotype through the production of IL-2. In vivo
experiments show that D3R expressed on OT-I lymphocytes favors a higher clonal
expansion and thereby an stronger protection against melanoma tumors.
The results obtained suggest that D3R promotes the formation of a more efficient
cytotoxic profile capable of producing higher percentages of IFN-γ and higher rates of
clonal expansion, which leads to greater survival in the face of a tumor challenge. / junio 2020
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Regulation of the Dopamine D3 Receptor by Adenylyl Cyclase 5Habibi Khorasani, Hedieh 10 May 2022 (has links)
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.
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