Rasd1 interacts with Ear2 (Nr2f6) to regulate renin transcription.
Tan, Jen Jen.
Ong, Angeline Shufen.
Date of Issue2011
School of Biological Sciences
The 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. Results: To 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, realtime 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. Conclusions: Our 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.
DRNTU::Science::Biological sciences::Molecular biology
BMC molecular biology
Copyright 2011 The Authors. The journal's website is located at http://www.biomedcentral.com/bmcmolbiol. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.