ISOLATION AND CHARACTERIZATION OF THE 5'-FLANKING REGION OF THE MOUSEGONADOTROPIN-RELEASING-HORMONE RECEPTOR GENE

The GnRH receptor (GnRH-R) is a cell surface, G protein-coupled recept or that is highly expressed in pituitary gonadotropes. Activation of t he receptor by GnRH stimulates the release of FSH and LH. Pituitary Gn RH-R numbers and, hence, gonadotrope responsiveness to GnRH vary under different conditions and are regulated to a large extent by GnRH itse lf. To study the transcriptional regulation of the GnRH-R gene, a geno mic clone containing 1.2 kilobases (kb) of the 5'-flanking region of m ouse GnRH-R gene was isolated and characterized. A major transcription al start site was identified 62 nucleotides up-stream of the translati onal start site by primer extension and ribonuclease protection analys es. The promoter region does not contain canonical TATA sequences in t he appropriate location. To determine whether this putative promoter i s functional, it was subcloned into a luciferase reporter plasmid (GnR H-RLuc), and its transient expression was studied in cell lines of gon adotrope (alpha T3-1) and somatolactotrope (GH(3)) origins as well as those of nonpituitary origin (JEG-3 and CV-1). Luciferase activity was increased in alpha T3-1 (246-fold +/- 34.5-fold; P < 0.005) compared with the promoterless vector control but was considerably lower in GH( 3) (41-fold +/- 3.9-fold; P < 0.005), JEG-3 (12-fold +/- 0.9-fold; P < 0.005) and CV-1 (8-fold +/- 1.3-fold) indicating that GnRH-RLuc is pr eferentially expressed in cells of gonadotrope origin. Furthermore, Gn RH agonist stimulated luciferase activity 3.4-fold +/- 0.3-fold (P < 0 .005) above basal levels in GH(3) cells cotransfected with rat GnRH-R complementary DNA, indicating that the GnRH-R promoter sequence is res ponsive to this ligand. In summary, we have identified and partially c haracterized the promoter region of the mouse GnRH-R and demonstrated that the regulatory elements for tissue-specific expression as well as for GnRH regulation are present within a 1.2-kb 5'-flanking region of the mouse GnRH-R gene.