| Abstract No.: | B-B2066 |
| Country: | Canada |
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| Title: | NEUROTENSIN REGULATES D2 DOPAMINE RECEPTOR INTERNALIZATION.
THIBAULT DOMINIC, PINEYRO FILPO GRACIELA, TRUDEAU LOUIS-ÉRIC. |
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| Authors/Affiliations: | 1 Dominic Thibault*; 1 Graciela Pineyro Filpo; 1 Louis-Éric Trudeau;
1 Université de Montréal,QC, Canada
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| Content: | Objectives: Neurotensin (NT) is a small neuropeptide that is very abundant in the brain. It is known to exert a potent excitatory effect on the dopaminergic system by binding to NTS1 receptors expressed in substantia nigra (SN) and ventral tegmental area (VTA) dopamine (DA) neurons. There is substantial evidence showing that NT reduces the ability of the DA D2 receptor (D2R) expressed by DA neurons to produce a negative feedback on the activity of these cells. Postsynaptic D2 receptors in the striatum are also negatively regulated by NT. This regulation of D2R function by NT is dependent on the activation of protein kinase C (PKC), a downstream element of the NTS1 signalling cascade. Because PKC can also trigger internalization of D2Rs, we hypothesize that the effect of NT on D2R function might involve regulation of D2R membrane expression and trafficking. To address this issue directly, we expressed these receptors in a heterologous system and studied their internalization.
METHODS: HEK 293 cells were transiently transfected with different combinations of a flag-tagged D2R (D2L; long isoform) and a HA-tagged NTS1. Agonist treatment consisted in the application of either or both 10 µM quinpirole (Quin, selective D2R agonist) and 10 nM NT. Membrane expression of transfected receptors was measured with an enzyme-linked immuno-sorbent assay (ELISA). Specific signal generated by surface immunoreactivity was obtained by subtracting unspecific labelling from non-transfected cells, and the final results were expressed as the percentage of membrane signal normalized to untreated transfected cells. RESULTS: In cells that co-expressed D2L and NTS1, Quin induced a rapid 18.50 ± 4.24 % reduction of membrane D2L which was maximal after 5 minutes and lasted for 60 minutes, whereas co-treatment with Quin plus NT increased this internalization to 33.87 ± 6.87 % (n = 4). Pre-treatment for 30 minutes with the PKC inhibitor chelerythrine (CHE) prevented the increase in internalization induced by NT (16.44 ± 12.81 % after 5 min; n = 2). NT did not affect the agonist-induced internalization of D2L in the absence of NTS1 and did not trigger D2L internalization when applied alone on cells with standard co-expression levels of D2L and NTS1. However, when the level of co-transfected NTS1 DNA was increased threefold relative to standard levels, stimulation with NT alone produced a 23.90 ± 7.74 % internalization of the D2L (n = 3). Conversely, co-activation of D2L did not affect the agonist-dependent internalization of NTS1, which was 64.33 ± 13.89 % after 5 minutes with NT alone, compared to 65.91 ± 3.69 % with Quin + NT co-treatment (n = 3).
CONCLUSIONS: In transfected HEK 293 cells, NTS1 activation causes an increase in agonist-dependent (through a PKC sensitive mechanism) and -independent internalization of the D2L. Our observation that D2L activation does not affect NTS1 internalization makes it unlikely that the D2-NTS1 interaction results from internalization of heteromeric receptor complexes. However, further experiments will be required to conclusively exclude this possibility. These data provide new insights in the |
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