Neurotensin Triggers Dopamine D2 Receptor Desensitization through a Protein Kinase C and β-Arrestin1-dependent Mechanism

Louis-Éric Trudeau,Paul R. Albert,Dominic Thibault,Graciela Pineyro

doi:10.1074/jbc.m110.166454

Abstract

The peptide neurotensin (NT) is known to exert a potent excitatory effect on the dopaminergic system by inhibiting D2 dopamine (DA) receptor (D2R) function. This regulation is dependent on activation of PKC, a well known effector of the type 1 NT receptor (NTR1). Because PKC phosphorylation of the D2R has recently been shown to induce its internalization, we hypothesized that NT acts to reduce D2R function through heterologous desensitization of the D2R. In the present study, we first used HEK-293 cells to demonstrate that NT induces PKC-dependent D2R internalization. Furthermore, internalization displayed faster kinetics in cells expressing the D2R short isoform, known to act as an autoreceptor in DA neurons, than in cells expressing the long isoform, known to act as a postsynaptic D2R. In patch clamp experiments on cultured DA neurons, overexpression of a mutant D2S lacking three key PKC phosphorylation sites abrogated the ability of NT to reduce D2R-mediated cell firing inhibition. Short interfering RNA-mediated inhibition of β-arrestin1 and dynamin2, proteins important for receptor desensitization, reduced agonist-induced desensitization of D2R function, but only the inhibition of β-arrestin1 reduced the effect of NT on D2R function. Taken together, our data suggest that NT acutely regulates D2 autoreceptor function and DA neuron excitability through PKC-mediated phosphorylation of the D2R, leading to heterologous receptor desensitization.

Highlights

We demonstrate that the effect of NT on D2R function in cultured DA neurons is strongly reduced by overexpression of a D2S receptor mutated on PKC phosphorylation sites that are critical for D2R desensitization and by siRNA-mediated downregulation of ␤-arrestin[1]
Our analysis showed a significant increase in the Pearson correlation coefficient of the Rab[5] and D2S signals, compared with control following a 10-min treatment with Quin (10 ␮M), NT (10 nM), or both; this can be interpreted as an increase in Rab5D2S colocalization (Ϫ0.052 Ϯ 0.019 for control, 0.203 Ϯ 0.032 for Quin, 0.115 Ϯ 0.041 for NT, and 0.201 Ϯ 0.049 for QuinϩNT)
In light of our results showing that NT triggered D2R internalization in transfected HEK-293 cells and our demonstration that mutation of phosphorylation sites important for PKC-dependent D2R internalization prevented the NTR1-D2R interaction in cultured DA neurons, we evaluated whether siRNA-mediated down-regulation of desensitization-related proteins (Fig. 4, A and B) would alter the physiological effect of NT on D2R function

Summary

Introduction

R. A.), and an infrastructure grant from the Fonds de la Recherche en Santedu Quebec to the Groupe de Recherche sur le Systeme Nerveux Central. To increase DA neuron excitability via distinct mechanisms, one of which consists in a reduction of presynaptic D2 autoreceptor function (5– 8). Activation of these D2Rs by DA normally causes cell hyperpolarization, which leads to a decrease in both spontaneous firing and DA release (9 –13). The goal of the present study was to assess whether NTR1 activation leads to PKC-induced D2R desensitization and to establish whether a mechanism of agonist-independent desensitization of the D2R could have any functional significance for the physiological effects of NT on DA neurons. We demonstrate that the effect of NT on D2R function in cultured DA neurons is strongly reduced by overexpression of a D2S receptor mutated on PKC phosphorylation sites that are critical for D2R desensitization and by siRNA-mediated downregulation of ␤-arrestin[1]

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Conclusion