Abstract
BackgroundThe Rasd1 protein is a dexamethasone induced monomeric Ras-like G protein that oscillates in the suprachiasmatic nucleus (SCN). Previous studies have shown that Rasd1 modulates multiple signaling cascades. However, it is still unclear exactly how Rasd1 carries out its function. Studying protein-protein interactions involving Rasd1 may provide insights into its biological functions in different contexts.ResultsTo further explore the molecular function of Rasd1, we performed a yeast two-hybrid screen and identified Ear2, a negative regulator of renin transcription, as an interaction partner of Rasd1. We validated the interaction in vitro and in transfected COS-7 cells. We further confirmed the interaction of endogenous Rasd1 and Ear2 from HEK293T cell and mouse brain extract. Rasd1 inhibited transcriptional repression by Ear2 on a renin promoter-luciferase reporter construct both in the presence and absence of all-trans-retinoic acid. Moreover, real-time RT-PCR showed upregulation of endogenous renin transcription in As4.1 cells over-expressing Rasd1. We demonstrated that the ligand binding domain of Ear2 is required for physical and functional interaction between the two proteins. In addition, we demonstrated that shRNA-mediated knockdown of Rasd1 results in further repression of Ear2-mediated renin transcription, whereas induction of Rasd1 by dexamethasone counteracts the effects of shRNA-mediated Rasd1 knockdown. Finally, our study showed that Rasd1 missense mutations not only attenuate their physical interaction with Ear2 but also abolish their ability to counteract repression of renin transcription mediated by Ear2.ConclusionsOur study provides evidence for physical and functional interactions between Rasd1 and Ear2. The results suggest that their interactions are involved in renin transcriptional regulation. These findings not only reveal a novel role for Rasd1-medated signaling but also provide the basis for potential intervention of renin expression.
Highlights
The Rasd1 protein is a dexamethasone induced monomeric Ras-like G protein that oscillates in the suprachiasmatic nucleus (SCN)
It was determined that Ear2 negatively regulates renin expression by competing with retinoic acid receptor/ retinoid X receptor (RAR/RXR) for binding to the retinoic acid response elements (RARE) on the renin enhancer [13]
Our results show that Rasd1 alleviates the Ear2mediated transcriptional repression of renin promoter activity in a dosage-dependent manner (Figure 2B)
Summary
The Rasd protein is a dexamethasone induced monomeric Ras-like G protein that oscillates in the suprachiasmatic nucleus (SCN). Previous studies have shown that Rasd modulates multiple signaling cascades. It is still unclear exactly how Rasd carries out its function. COUP-TFs have been shown to bind to a number of variable direct and indirect repeats with different spacings between the repeats [15] to affect a large plethora of genes [16,17,18,19]. Nuclear hormone receptors have the ability to bind directly to DNA and regulate the expression of specific target genes; they are extremely crucial to the development, homeostasis and metabolism of an organism [16,20,21, 23,24,25]. Ear knockout mice are viable and fertile, but they possess circadian and nociception defects and abnormal locus coeruleus (LC) development [25]
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