Endocrinology, Vol 135, 2300-2306, Copyright © 1994 by Endocrine Society

ARTICLES

Isolation and characterization of the 5'-flanking region of the mouse gonadotropin-releasing hormone receptor gene

CT Albarracin, UB Kaiser and WW Chin
Department of Medicine, Brigham and Women's Hospital, Howard Hughes Medical Institute, Boston, Massachusetts 02115.

The GnRH receptor (GnRH-R) is a cell surface, G protein-coupled receptor that is highly expressed in pituitary gonadotropes. Activation of the receptor by GnRH stimulates the release of FSH and LH. Pituitary GnRH-R numbers and, hence, gonadotrope responsiveness to GnRH vary under different conditions and are regulated to a large extent by GnRH itself. To study the transcriptional regulation of the GnRH-R gene, a genomic clone containing 1.2 kilobases (kb) of the 5'-flanking region of mouse GnRH-R gene was isolated and characterized. A major transcriptional start site was identified 62 nucleotides upstream of the translational start site by primer extension and ribonuclease protection analyses. The promoter region does not contain canonical TATA sequences in the appropriate location. To determine whether this putative promoter is functional, it was subcloned into a luciferase reporter plasmid (GnRH-RLuc), and its transient expression was studied in cell lines of gonadotrope (alpha T3-1) and somatolactotrope (GH3) 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 GH3 (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 preferentially expressed in cells of gonadotrope origin. Furthermore, GnRH agonist stimulated luciferase activity 3.4- fold +/- 0.3-fold (P < 0.005) above basal levels in GH3 cells cotransfected with rat GnRH-R complementary DNA, indicating that the GnRH-R promoter sequence is responsive to this ligand. In summary, we have identified and partially characterized 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.

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