Figure 4

Ear2 ligand binding domain interacts with Rasd1. (A) Schematic diagram showing the truncated constructs of Ear2. Ear2 contains 4 main domains: an activator function I site, a DNA binding domain, a zinc finger domain and a ligand binding domain. Full length (FL) Ear2 has 390 amino acids and each of the six truncated Ear2 constructs is represented by their amino acid numbers on the left. (B) Immunoblot analysis showed that Rasd1 binds to Ear2 ligand binding domain. COS-7 cells were co-transfected with pHisHA-Rasd1 and the indicated pGST-Ear2 constructs. HisHA-Rasd1 from the cell lysates was immobilized on Ni-NTA beads and GST-Ear2 constructs bound to the complexes were eluted by heating at 95°C for 10 minutes, and detected by immunoblotting with anti-GST (lanes 1 and 5-7). GST-Ear2 constructs not co-precipitated with HisHA-Rasd1 were detected in the flow-through (lanes 2-4 and 8). (C) Luciferase study showed that Ear2 ligand binding domain and DNA binding domain are required for interacting with Rasd1 to modulate renin transcription. COS-7 cells were transfected with p4.1-Luc (2.0 μg), pGST-Ear2 (1.5 μg) or plasmids expressing Ear2 truncated constructs and pHisHA-Rasd1 (1.5 μg) as indicated, together with pSV-β-gal (0.5 μg). Total DNA transfected was held constant with the respective carrier vector plasmids. Controls are transfected with pGL3-basic (2.0 μg), pSV-β-gal (0.5 μg) and appropriate amounts of the respective carrier vectors. Relative luciferase activity was normalized against β-gal activity. Ear2 constructs that were missing their DNA binding domains (Ear2-N53, Ear2-C131, Ear2-C194) did not significantly repress renin transcription. Rasd1 alleviated Ear2-repressed renin transcription only if Ear2 ligand binding domain is present (Ear2-FL, Ear2-C54). * denotes p < 0.05.