Involvement of upstream open reading frames in regulation of rat V1bvasopressin receptor expression

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Abstract
The V1bvasopressin 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 ∼5 kb from the translation start site), and characterized the 5′-flanking region of the rat V1breceptor 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 V1bexpression. 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 V1breceptor 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 V1bexpression is functionally linked to any intracellular and/or extracellular factor(s) awaits further research.