Involvement of upstream open reading frames in regulation of rat V_1bvasopressin receptor expression

Abstract

The V(1b) vasopressin receptor, expressed mainly in the corticotroph of the anterior pituitary, mediates the stimulatory effect of vasopressin on ACTH release. To clarify the regulation of receptor expression, we cloned, sequenced (up to approximately 5 kb from the translation start site), and characterized the 5'-flanking region of the rat V(1b) receptor gene. We identified the transcription start site by amplification of cDNA ends and found a new intron within the 5'-untranslated region (5'-UTR) by comparing the sequence with that of cDNA. We then confirmed that the obtained promoter indeed has transcriptional activity by use of the luciferase reporter in AtT-20 mouse corticotroph cells. Interestingly, there were five short upstream open reading frames (uORFs) located within the 5'-UTR that were found to suppress V(1b) expression. Subsequent mutational analyses showed that the two downstream uORFs have an inhibitory effect on expression in both homologous and heterologous contexts. Furthermore, the inhibition did not accompany a parallel decrease in mRNA, suggesting that the suppressive effect occurs at a level downstream of transcription. Taken together, our data strongly suggest that the expression of the V(1b) receptor is regulated at the posttranscriptional as well as transcriptional level through uORFs within the 5'-UTR region of the mRNA. Whether the uORF-mediated regulation of V(1b) expression is functionally linked to any intracellular and/or extracellular factor(s) awaits further research.

Recognition of a Novel Stage of Betaherpesvirus Latency in Human Herpesvirus 6

“…1), were considered to be translated into the IE1 protein at this novel, intermediate stage. For some mRNAs that have small uORFs that restrict the translation of the downstream ORFs, it has been reported that modulation of both translational regulation and the mRNA level is important for release from the uORF control (14,28). An alteration in the regulation of translation as well as the increase of the H6LTs might have contributed to the increased IE1 protein expression (Table 2).…”

Transcriptional Regulation of the Pituitary Vasopressin V1b Receptor Involves a GAGA-binding Protein

“…To study the regulation of the V1bR, we (10) and others (9) have recently isolated and characterized the 5Ј-flanking region of the rat V1bR. Computer analysis of the putative promoter region (bp Ϫ2161 to ϩ377, corresponding to 2.1 kb of the putative promoter, exon 1, intron 1, and 164 bp of exon 2) showed no TATA box, but several potential sites for glucocorticoid, AP1, and cAMP regulation.…”

Transcriptional and post-transcriptional mechanisms regulating the rat pituitary vasopressin V1b receptor gene

“…This DNA fragment shows promoter activity in the hypothalamic cell line, H32 (Volpi et al 2002) and in MCF-7 cells which express endogenous V1b receptor, as well as in cells not expressing the receptor such as in COS-7, CHO and PC12 cell lines (Rabadan-Diehl et al 2000). Promoter activity in the pituitary tumor cell line AtT20, however, remains controversial (Rabadan-Diehl et al 2000, Nomura et al 2001. A construct containing a 2161 to +72 fragment, without the intron and distal sequence, has no promoter activity in any cell line tested, suggesting that intron 1 is important for receptor gene activation (Rabadan-Diehl et al 2000, Volpi et al 2002.…”

“…increases promoter activity without changes in transfection efficiency, strongly suggesting that the region between bp 294 and 2161 of the V1bR promoter contains repressor elements (Nomura et al 2001, Volpi et al 2002. The genomic sequences responsible for tissue-specific expression of the V1b receptors have yet to be elucidated.…”

“…This suggests that V1bR content in the pituitary does not primarily depend on the levels of V1bR mRNA but on the efficiency of mRNA translation into protein and post-translational events. Recent studies have focused on the translational regulation of the V1bR and especially on the role of the 5 -UTR , Nomura et al 2001. The rat V1bR 5 -UTR is long, structurally complex, and displays several small open reading frames (ORF) upstream of the initiating methionine (uORF), features likely to reduce the ability of ribosomes to linearly scan the V1b 5 -UTR mRNA according to conventional scanning models ( Fig.…”