Cell-specific expression of the mouse gonadotropin-releasing hormone (GnRH) receptor gene is conferred by elements residing within 500 bp of proximal 5′ flanking region

Abstract

Gonadotropin-releasing hormone (GnRH) is a decapeptide produced by the hypothalamus. Upon binding to specific high-affinity receptors on gonadotrope cells of the anterior pituitary gland, GnRH stimulates the synthesis and secretion of LH. In light of the critical role of GnRH in reproduction much effort has been directed toward understanding the regulation of this hormone and its cognate receptor. The recent availability of genomic clones for the GnRH receptor has facilitated research to address the molecular mechanisms underlying regulation of GnRH receptor gene expression. We have expanded the analysis of the promoter for the mouse GnRH receptor gene and report that in addition to transcriptional start sites located within 100 bp of the translation start codon there is a more distal transcriptional start site approximately 200 bp 5' of the initiation codon. The initiation of transcription from this more distal site was sufficient to confer cell-specific expression on luciferase. Further, transient expression assays of constructs containing progressive 5' deletions in the GnRH receptor gene promoter reveal the presence of one or morecis-acting elements located between -500 and -400 (relative to ATG) necessary for transcriptional activity in the gonadotrope-derived αT3 cell line. Finally, αT3 but not COS-7 cell nuclear extract contained protein(s) that bind to at least two separate motifs contained within the -500 to -400 region. We suggest that activation of GnRH receptor gene expression in the αT3 cell line requires the binding of at least two transcriptional regulatory proteins to basal enhancer elements located within a 100 bp region between -500 to -400 relative to the translation start codon in the mouse GnRH receptor gene.

Identification and Characterization of the Gonadotropin-releasing Hormone Response Elements in the Mouse Gonadotropin-releasing Hormone Receptor Gene

“…Functional analysis by transient transfection of ␣T3-1 cells with the 1.2-kb 5Ј-flanking region of the mGnRHR gene (Ϫ1164/ϩ62 [ Figs. 2 and 3]) confirmed previous observations (13,25) that this region contains an element(s) that is necessary for tissue-specific basal expression as well as for GnRH responsiveness. By using deletion and mutational analysis in ␣T3-1 cells, Duval et al (26) recently identified a tripartite enhancer that appears to be responsible for regulating cell-specific basal expression of the GnRHR gene.…”

“…9B). These findings were not unexpected given that no cAMP-response element-like sequence has been identified in the mGnRHR gene (13,25), although there may be cAMP-response element-like elements in the rat and human GnRHR genes (44,45). Taken together, these data suggest that the response of the mGnRHR gene to GnRHAg stimulation is mediated via PKC and not PKA.…”

“…This region has also been used in transgenic mice to show that it is sufficient to mediate gonadotrope-specific expression in vivo (24). Preliminary studies on the 5Ј-flanking putative promoter region of the mouse, human, and sheep GnRHR genes reveal complex organization with multiple TSS that are occasionally associated with TATA boxes (13,25). In the mGnRHR gene, the major TSS was shown to be located 62 nucleotides upstream of the translational start site by primer extension and ribonuclease protection analysis of ␣T3-1 gonadotrope mRNA (13).…”

Steroidogenic Factor-1 Interacts with a Gonadotrope-Specific Element within the First Exon of the Human Gonadotropin-Releasing Hormone Receptor Gene to Mediate Gonadotrope-Specific Expression

“…In fact, the presence of SF-1 at multiple levels of the hypothalamic-pituitary-gonadal axis (15)(16)(17) further suggests the regulatory role of SF-1 as a master protein directing its effect at multiple levels of the reproductive axis. In the mouse GnRHR gene, it has been shown that the proximal 500 bp relative to the ATG site with two putative GSE motifs is crucial to the gonadotrope-specific promoter activity (18,5). More recently, the GSE site residing at Ϫ250 to Ϫ232 was identified to be one of the cis-elements within a tripartite tissue-specific enhancer to confer gonadotrope-specific expression of the murine GnRHR gene (9,19).…”

“…On the other hand, based on the initial characterization of the rat, mouse, human, and ovine GnRHR genes, the 5Ј ends of these genes were found to be structurally different from each other. The human and ovine GnRHR genes contain multiple CAP sites with over 650 and 800 bp of 5Ј-untranslating region (5Ј-UTR), respectively (4,3), whereas the murine and rat GnRHR genes are comprised of less than 200 bp of 5Ј-UTR (5,6). The structural differences in the mammalian GnRHR 5Ј-flanking regions suggest that they are regulated in different manners, and there is still no information on the transcriptional regulation of the hGnRHR to date.…”