Abstract
The D1 dopamine receptor subtype is expressed in the brain, kidney and lymphocytes. D1 receptor function has been extensively studied and the receptor has been shown to modulate a wide range of physiological functions and behaviors. The expression of D1 receptor is known to change during development, disease states and chronic treatment; however, the molecular mechanisms that mediate the changes in D1 receptor expression under these circumstances are not well understood. While previous studies have identified extracellular factors and signaling mechanisms regulating the transcription of D1 receptor gene, very little is known about other regulatory mechanisms that modulate the expression of the D1 receptor gene. Here we report that the D1 receptor is post-transcriptionally regulated during postnatal mouse brain development and in the mouse CAD catecholaminergic neuronal cell line. We demonstrate that this post-transcriptional regulation is mediated by a molecular mechanism involving noncoding RNA. We show that the 1277 bp 3′untranslated region of D1 receptor mRNA is necessary and sufficient for mediating the post-transcriptional regulation. Using deletion and site-directed mutagenesis approaches, we show that the D1 receptor post-transcriptional regulation is specifically mediated by microRNA miR-142-3p interacting with a single consensus binding site in the 1277 bp 3′untranslated region of D1 receptor mRNA. Inhibiting endogenous miR-142-3p in CAD cells increased endogenous D1 receptor protein expression levels. The increase in D1 receptor protein levels was biologically significant as it resulted in enhanced D1 receptor-mediated signaling, determined by measuring the activation of both, adenylate cyclase and, the dopamine- and cAMP-regulated phosphoprotein, DARPP-32. We also show that there is an inverse correlation between miR-142-3p levels and D1 receptor protein expression in the mouse brain during postnatal development. This is the first study to demonstrate that the post-transcriptional regulation of D1 receptor expression is mediated by microRNA-induced translational suppression.
Highlights
The neurotransmitter dopamine binds and activates two major subfamilies of dopamine receptors
There is no significant difference in the expression of D1 receptor mRNA between day of birth (P0) through postnatal day 14 (P14) (Fig. 1A and 1C); in contrast, the relative expression of D1 receptor protein is low at P0 and begins to increase at P7 with a significant increase in expression on P14 (Fig. 1B and 1D)
The results presented in this paper establishes post-transcriptional regulation as a significant mechanism that controls the expression of D1 dopamine receptor protein during postnatal mouse brain development (Fig. 1) and in the mouse CAD catecholaminergic cell line (Fig. 2)
Summary
The neurotransmitter dopamine binds and activates two major subfamilies of dopamine receptors. Several studies have shown that the D1 dopamine receptor is expressed in the kidneys [2] and lymphocytes [3]. D1 receptor signaling function has been extensively studied and it has been shown to activate adenylate cyclase and modulate ion channel function [4]. D1 receptors modulate the sodium-potassium ATPase and the sodium-hydrogen exchanger and regulate diuresis and natriuresis [5]. Peripheral dopamine acting via D1 receptor expressed on antigenpresenting dendritic cells and T-cells was shown to modulate the differentiation of various types of T-cells following immune activation [3]. The D1 receptor is expressed in the periphery and the brain and plays an important role in many physiological and pathophysiological conditions
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