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) recept or was isolated, and its structural organization and promoter region w ere characterized. The gene was found to consist of three exons that e ncode the receptor protein, and spanned about 20 kb. Of two genomic cl ones 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, respect ively. The first intron is at least 12 kb in length and is located bet ween 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 7 40, separating the fifth and sixth transmembrane domains. Genomic blot s in combination with cloning and sequencing suggested that a single G nRH receptor gene is present in the rat genome. Primer extension indic ated that the transcription start site is located 103 nt upstream of t he 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-I 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 w ere fused to the luciferase reporter gene and transiently expressed in immortalized pituitary gonadotrophs (alpha T3-1 cells) and hypothalam ic neurons (GT1-7 cells), and in nonpituitary (COS-7) cells. Luciferas e gene expression was significantly increased by all three fragments i n pituitary and hypothalamic cells, but not in COS-7 cells. The promot er activity of the 1.2 kb fragment was higher than that of the other f ragments. Forskolin and cAMP analogs increased luciferase gene express ion in both alpha T3-1 and GT1-7 cells, but activation of protein kina se C by phorbol myristate acetate had no effect. These studies indicat e that positive and negative regulatory elements are present within th e 1.8 kb 5' flanking sequence of the GnRH receptor. Knowledge of the g enomic organization and analysis of the promoter region of the rat GnR H receptor gene will facilitate the elucidation of its transcriptional control in pituitary gonadotrophs and hypothalamic neurons. (C) 1997 Elsevier Science Ireland Ltd.