Structural organization and characterization of the promoter region of the rat gonadotropin-releasing hormone receptor gene.

The gene encoding the rat gonadotropin-releasing hormone (GnRH) receptor was isolated, and its structural organization and promoter region were characterized. The gene was found to consist of three exons that encode the receptor protein, and spanned about 20 kb. Of two genomic clones analyzed, one contained the 5'-untranslated region and the first exon, and the other contained the second and third exons. The sizes of the first, second, and third exons are 625, 217, and 1476 nt, respectively. The first intron is at least 12 kb in length and is located between nucleotides 522 and 523 of the cDNA reading frame, in the middle of the fourth transmembrane domain. The second intron is about 2.5 kb and is also located in the reading frame between nucleotides 739 and 740, separating the fifth and sixth transmembrane domains. Genomic blots in combination with cloning and sequencing suggested that a single GnRH receptor gene is present in the rat genome. Primer extension indicated that the transcription start site is located 103 nt upstream of the translational start codon. A putative TATA box is positioned 23 nt in front of the transcription initiation site. The 1.8 kb 5' flanking sequence contains an SF-1 site, an AP-1 site, CCAAT sequences, a Pit-1 binding site, and a potential CRE-like sequence. To evaluate promoter activity, the 1.8 kb and two 5' deleted fragments of 1.2 and 0.6 kb were fused to the luciferase reporter gene and transiently expressed in immortalized pituitary gonadotrophs (alphaT3-1 cells) and hypothalamic neurons (GT1-7 cells), and in nonpituitary (COS-7) cells. Luciferase gene expression was significantly increased by all three fragments in pituitary and hypothalamic cells, but not in COS-7 cells. The promoter activity of the 1.2 kb fragment was higher than that of the other fragments. Forskolin and cAMP analogs increased luciferase gene expression in both alphaT3-1 and GT1-7 cells, but activation of protein kinase C by phorbol myristate acetate had no effect. These studies indicate that positive and negative regulatory elements are present within the 1.8 kb 5' flanking sequence of the GnRH receptor. Knowledge of the genomic organization and analysis of the promoter region of the rat GnRH receptor gene will facilitate the elucidation of its transcriptional control in pituitary gonadotrophs and hypothalamic neurons.