Involvement of upstream open reading frames in regulation of rat V-1b vasopressin receptor expression

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, seque nced (up to similar to5 kb from the translation start site), and characteri zed 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 int ron within the 5'-untranslated region (5'-UTR) by comparing the sequence wi th that of cDNA. We then confirmed that the obtained promoter indeed has tr anscriptional activity by use of the luciferase reporter in AtT-20 mouse co rticotroph cells. Interestingly, there were five short upstream open readin g 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 hetero logous contexts. Furthermore, the inhibition did not accompany a parallel d ecrease in mRNA, suggesting that the suppressive effect occurs at a level d ownstream 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 funct ionally linked to any intracellular and/or extracellular factor(s) awaits f urther research.