MULTIPLE ELEMENTS IN THE DISTAL PART OF THE 1.2 KB 5'-FLANKING REGIONOF THE RAT GNRH RECEPTOR GENE REGULATE GONADOTROPE-SPECIFIC EXPRESSION CONFERRED BY PROXIMAL DOMAIN

Several lines of evidence indicate that the number of GnRH receptors ( GnRH-R) and therefore, gonadotrope responsiveness to GnRH, is highly d ependent upon the level of GnRH-R mRNA. To explore this aspect of regu lation, we have isolated a 3.3 kb fragment encompassing the promoter r egion of the rat GnRH-R gene. Primer extension and RNase protection as says allowed the identification of five major transcriptional start si tes located within the 110 bp region upstream of the translation start codon. Transfection experiments using the CAT reporter gene demonstra ted that the 1261 bp 5' flanking region is required to direct high eff icient expression in the gonadotrope-derived alpha T3-1 cell line thus contrasting with mouse in which the only 500 bp proximal sequence app eared to be sufficient. Another difference between Pat and mouse was a pparent in the 183 bp region of the rat promoter which induced a 3-fol d stimulation of thymidine kinase promoter activity in both alpha T3-1 and CHO cells. Subsequent deletion analysis of the region residing be tween - 1261 and -519 revealed the presence of multiple regulatory dom ains that contributed to the cell-specific activity. However, despite this efficiency in the context of the wild-type promoter, they failed to induce the activity of the minimal thymidine kinase (TK) promoter i n the absence of the proximal 600 bp promoter region. Accordingly, a c omposite TK promoter containing the entire 1.2 kb promoter induced a 1 0-fold increase in the activity of the TK promoter in alpha T3-1 cells . Taken together these data suggest that distal regulatory regions are critical and require cooperation with proximal specific-promoter elem ents for activating basal R-GnRH gene expression in gonadotrope cells. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.