Rasd1 interacts with Ear2 (Nr2f6) to regulate renin transcription.
BACKGROUND
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, 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.


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.
School of Biological Sciences, Department of Genomics and Genetics, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
Jen Jen
JJ
Tan
Shufen Angeline
SA
Ong
Ken-Shiung
KS
Chen
D000818:Animals; D019556:COS Cells; D051836:COUP Transcription Factors / Q000235:genetics*; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D034741:RNA, Small Interfering / Q000378:metabolism; D011987:Receptors, Steroid / Q000235:genetics*; D012083:Renin / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014158:Transcription, Genetic; D014212:Tretinoin / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D018631:ras Proteins / Q000235:genetics*
D051836:COUP Transcription Factors; D004268:DNA-Binding Proteins; C075835:NR2F6 protein, human; C484221:Nr2f6 protein, mouse; C400132:RASD1 protein, human; D034741:RNA, Small Interfering; D011987:Receptors, Steroid; D012097:Repressor Proteins; D014212:Tretinoin; D012083:Renin; C498511:Rasd1 protein, mouse; D018631:ras Proteins
Science. 1989 Jan 27;243(4890):542-4
2563177
Neurobiol Aging. 2001 Jul-Aug;22(4):541-6
11445253
Genes Dev. 2005 Mar 1;19(5):614-25
15741322
Ann Intern Med. 1979 Jan;90(1):43-7
420460
Arterioscler Thromb Vasc Biol. 2010 Aug;30(8):1529-34
20631353
Lancet. 1979 Jun 9;1(8128):1213-5
87679
Nature. 1996 Aug 29;382(6594):810-3
8752274
Crit Rev Neurobiol. 1998;12(4):305-17
10348613
Physiology (Bethesda). 2008 Aug;23:187-93
18697992
Hypertension. 2005 Jan;45(1):3-8
15545507
Ann N Y Acad Sci. 2000 Apr;903:437-41
10818534
Neurosci Biobehav Rev. 2002 Aug;26(5):529-52
12367589
Mol Cell Biol. 2000 Apr;20(7):2604-18
10713182
J Appl Physiol (1985). 2009 Dec;107(6):1965-71
19628724
Endocr Regul. 2001 Jun;35(2):65-70
11563933
Neuroscience. 1999;93(3):877-84
10473253
Mol Cell Biol. 1988 Aug;8(8):3235-43
3145408
Mol Cell Biol. 1996 May;16(5):2332-40
8628300
Semin Cell Dev Biol. 2006 Jun;17(3):345-51
16765612
Cell. 1995 Dec 15;83(6):835-9
8521507
Eur J Pharmacol. 1991 Aug 6;200(2-3):289-92
1664329
Br J Clin Pharmacol. 1979;7 Suppl 2:205S-211S
223614
Biochemistry. 1995 Mar 14;34(10):3470-7
7880841
J Biol Chem. 1990 Nov 15;265(32):19916-22
2174057
Eur J Pharmacol. 1996 Apr 4;300(1-2):113-7
8741175
Hypertension. 2006 Mar;47(3):461-6
16446393
Am J Physiol. 1996 Jul;271(1 Pt 2):F94-100
8760248
Proc Natl Acad Sci U S A. 1981 Nov;78(11):6940-3
6273896
J Neurol Sci. 2002 Jul 15;199(1-2):25-9
12084438
Mol Cell Biol. 1997 Mar;17(3):1129-43
9032240
J Biol Chem. 2008 Dec 12;283(50):34728-37
18922798
Annu Rev Biochem. 1993;62:851-91
8352603
Neuron. 2000 Oct;28(1):183-93
11086993
Endocrinology. 2001 Jun;142(6):2631-40
11356714
Curr Protein Pept Sci. 2005 Aug;6(4):355-71
16101434
Gene. 1989 Apr 15;77(1):51-9
2744487
Cell. 2002 Jul 12;110(1):107-17
12151001
Physiol Rev. 2001 Jul;81(3):1269-304
11427696
Psychoneuroendocrinology. 1986;11(3):277-94
3538110
Sci STKE. 2004 Sep 07;2004(250):RE13
15367757
J Biol Chem. 1996 Apr 5;271(14):8196-202
8626511
Brain Res Mol Brain Res. 2003 Jan 31;110(1):1-6
12573527
Eur J Biochem. 1995 Feb 15;228(1):184-90
7883002
Hypertension. 1999 Mar;33(3):900-5
10082506
Methods Enzymol. 2002;350:87-96
12073338
J Mol Endocrinol. 1997 Oct;19(2):163-72
9343308
Ann Intern Med. 1979 Jan;90(1):19-23
217289
Proc Natl Acad Sci U S A. 1987 Apr;84(7):1814-8
3104905
Circ Res. 1999 Sep 17;85(6):479-88
10488050
Adv Physiol Educ. 2009 Dec;33(4):270-4
19948673
J Biol Chem. 2001 Feb 2;276(5):3597-603
11058598
Mol Cell Biol. 1992 Sep;12(9):4153-63
1324415
Intern Med. 1999 Apr;38(4):315-23
10361903
Oncogene. 1994 Sep;9(9):2691-8
8058333
Neurosci Biobehav Rev. 1994 Spring;18(1):21-53
8170622
Endocr Rev. 1997 Apr;18(2):229-40
9101138
Mol Endocrinol. 1990 Mar;4(3):375-83
2188116
Nucleic Acids Res. 1988 Dec 9;16(23):11057-74
2905047
Mech Dev. 1994 Jul;47(1):81-97
7947324
Cell. 1992 Oct 2;71(1):169-80
1394429
Cell. 1995 Dec 15;83(6):859-69
8521510
J Biol Chem. 1996 Sep 13;271(37):22499-505
8798416
J Biol Chem. 2001 Dec 7;276(49):45530-8
11564732
J Biol Chem. 1998 Feb 6;273(6):3129-31
9452419
Hypertension. 2001 Sep;38(3):332-6
11566900
Circ Res. 1997 Oct;81(4):558-66
9314837
Proc Natl Acad Sci U S A. 2005 Feb 22;102(8):3111-6
15710878
J Biol Chem. 1992 Aug 5;267(22):15849-60
1639815
Circ Res. 2003 May 16;92(9):1033-40
12690040
J Biol Chem. 2000 Jan 28;275(4):2763-70
10644740