Orexins (hypocretins), acting via their receptors, are involved in the control of feeding behaviour, sleep, arousal and energy homoeostasis. However, regulation of the human orexin receptor 2 (hOX2R) gene remains unknown. We have identified four transcripts arising from alternative splicing from three exons. These exon 1 variants were designated exons 1A, 1B and 1C on the basis of their 5′–3′ order. RT (reverse transcription)–PCR demonstrates the differential expression in various human tissues. The alternative 5′-UTRs (untranslated regions) possessed by these isoforms have different translational efficiencies, which regulate the level of protein expression. In the present study, we have demonstrated that the hOX2R gene is regulated by two promoters and the novel transcripts are regulated by the distal promoter located upstream of exon 1A. We have demonstrated that the AP-1 (activator protein 1) motif is critical for sustaining the basal activity of distal promoter. Analysis of the proximal promoter revealed the region regulating promoter activity contained putative binding elements including those for CREB (cAMP-response-element-binding protein), GATA-2 and Oct-1. Using the chromatin immunoprecipitation assay, we demonstrated that CREB, GATA-2 and Oct-1 transcription factors bind to these critical regulatory promoter elements. Mutational studies suggested that these motifs functioned independently, but have a compound effect regulating hOX2R gene transcription. Furthermore, proximal promoter activity is enhanced by both PKA (protein kinase A) and PKC (protein kinase C) pathway activation, via binding of CREB and GATA-2 transcription factors. In conclusion, we have demonstrated that expression of hOX2R is regulated by a complex involving a proximal PKA/PKC-regulated promoter and a distal promoter regulating tissue-specific expression of alternative transcripts which in turn post-transcriptionally regulate receptor levels.
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Research Article|
April 14 2010
Genomic organization and regulation of the human orexin (hypocretin) receptor 2 gene: identification of alternative promoters
Jing Chen;
Jing Chen
1Endocrinology and Metabolism Group, Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry CV4 7AL, U.K.
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Harpal S. Randeva
Harpal S. Randeva
1
1Endocrinology and Metabolism Group, Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry CV4 7AL, U.K.
1To whom correspondence should be addressed (email [email protected]).
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Publisher: Portland Press Ltd
Received:
November 12 2009
Revision Received:
February 10 2010
Accepted:
February 15 2010
Accepted Manuscript online:
February 15 2010
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 427 (3): 377–390.
Article history
Received:
November 12 2009
Revision Received:
February 10 2010
Accepted:
February 15 2010
Accepted Manuscript online:
February 15 2010
Citation
Jing Chen, Harpal S. Randeva; Genomic organization and regulation of the human orexin (hypocretin) receptor 2 gene: identification of alternative promoters. Biochem J 1 May 2010; 427 (3): 377–390. doi: https://doi.org/10.1042/BJ20091755
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