Abstract
The role of CT repeats (inverted GAGA box) in the rat vasopressin V1b receptor (V1bR) promoter in the transcriptional regulation of this gene was studied in H32 hypothalamic cells, which express endogenous V1bR. Transfection of a 2.5-kb V1bR fragment (2161 bp upstream and 377 bp downstream of the proximal transcriptional start point) into a luciferase vector (V1bRp2.5-Luc) results in promoter activity in these cells. The 670-bp proximal promoter fragment containing the GAGA box showed maximal promoter activity, whereas deletion of the GAGA box abolished transcription. Drosophila GAGA-binding protein increased V1bR promoter activity by 11-fold when cotransfected with V1bRp2.5-Luc and increased endogenous V1bR expression. Electrophoretic mobility shift assay showed specific binding of pituitary nuclear extracts to radiolabeled GAGA oligonucleotides, which increased following restraint stress in rats, a condition associated with V1bR up-regulation. DNA-binding activity involved a protein complex because it was abolished by deoxycholate. Size-exclusion column chromatography showed a complex of 127 kDa, which dissociated into approximately 70-kDa components after deoxycholate/Nonidet P-40 treatment. This study demonstrates that interactions of GAGA-binding proteins with the GAGA box of the V1bR promoter activate V1bR gene expression and provides a potential mechanism for physiological regulation of V1bR transcription.
