D1 Receptor Expression Is Regulated by MicroRNA Figure 7. D1 receptor post-transcriptional regulation is mediated by D1 receptor 39UTR and miR-142-3p even in the presence of a heterologous 39UTR polyadenylation signal. (A) Schematic representation of the parent pcDNA- b-gal construct with the bovine growth hormone (BGH) 39UTR and polyadenylation signal. Also shown is the recombinant pcDNA- b-gal (D1 39UTR 1277) construct in which the 1277 bp D1 receptor 39UTR was introduced between the stop codon of the b-galactosidase gene and the heterologous BGH 39UTR. (B) The parent pcDNA- b-gal construct, the wild type pcDNA- b-gal (D1 39UTR 1277) construct or pcDNA- b-gal (D1 39UTR 1277) constructs with mutations in the binding site for miR-10a*, miR-142-3p or miR-294/295 were transiently-transfected into non-differentiated CAD cells and 48 hours later b-galactosidase reporter activity was measured and normalized to total protein. The bars represent the mean values 6 s.e.m. (n = 6). *P,0.001, the level of b-galactosidase reporter activity was significantly increased in cells transfected with the pcDNA- b-gal (D1 39UTR 1277) construct with the miR-142-3p binding site mutated when compared to cells transfected with wild type pcDNA- b-gal (D1 39UTR 1277) or the other two mutant constructs, One Way ANOVA, post hoc SNK test. doi:10.1371/journal.pone.0049288.g007 versus 0.142 nM for control and miRZipTM anti 142-3p, respectively) suggesting that the inhibition of miR-142-3p specif- ically affected Emax. To further assess the biological significance of miR-142-3p regulation of D1 receptor expression, we evaluated the ability of D1 receptors to activate DARPP-32 in CAD cells transfected with miRZipTM anti 142-3p or empty miRZipTM Figure 8. Inhibition of miR-142-3p increases endogenous D1 receptor protein expression in CAD cells. Representative western blot (A) and cumulative data (B) showing the levels of D1 receptor protein normalized to total protein in non-differentiated (ND) and differentiated (D) CAD cells 48 hours post-transient transfection with 30 nM per well (in 12-well plate) FAM-labeled negative control anti mir (FAM Anti-mir) or mmu-miR- 142-3p anti-mir. The D1 receptor undergoes glycosylation and the three bands in the gel different glycosylated forms of the receptor. The bars represent the mean values 6 s.e.m. (n = 3). The endogenous D1 receptor protein level was significantly increased in non-differentiated (*, P,0.006) and differentiated (*, P,0.011) CAD cells transfected with miR-142-3p anti-mir compared to cells transfected with control FAM Anti-mir (two-tailed pair-wise Student’s t-test). doi:10.1371/journal.pone.0049288.g008 PLOS ONE | www.plosone.org 11 November 2012 | Volume 7 | Issue 11 | e49288