REGULATION OF GNRH RECEPTOR GENE EXPRESSION

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Gonadotropin-releasing hormone (GnRH) is synthesized and secreted from neuroendocrine cells of the hypothalamus. Upon binding to specific receptors located on gonadotropes of the anterior pituitary, GnRH not only stimulates but is obligatory for the s synthesis and secretion of LH and, to a lesser extent, FSH. In light of the pivotal role of GnRH in controlling reproductive function of mammals much effort has been devoted toward understanding the physiological consequences of regulation of this hormone and its's cognate receptor. For example, ovulation in mammals is dependent on a transitory increase in the secretory rate of LH that, in turn, results from both an increase in GnRH secretion and an increase in pituitary concentration of GnRH receptors. Recent availability of cDNA clones encoding the GnRH receptor has permitted direct measurement of mRNA for the GnRH receptor and researchers are beginning to establish the physiological importance of transcriptional mechanisms in mediating changes in the number of GnRH receptors. However, lack of genomic clones for the GnRH receptor has precluded any analysis of molecular mechanisms underlying transcriptional regulation of the GnRH receptor gene. Herein, we report the isolation of a partial genomic clone for the murine GnRH receptor and preliminary characterization of a cell-specific promoter located int he 5' flanking region. Thus, we are poised to begin a systematic analysis of the cis-acting DNA elements and trans-acting factors responsible for cell- specific and hormonally mediated expression of the GnRH receptor gene. Our long-term goals are to define the molecular mechanisms underlying both tissue-specific and hormonally-regulated expression of the GnRH receptor gene. Accordingly, in Specific Aim 1, we will use transient expression assays in alphaT3 cells, DNA-protein binding assays, and liposome-mediated gene transfer to study the requirements for cell-specific expression of the mouse GnRH receptor gene. In Specific Aim 2, we will use site-directed mutagenesis and transient expression assays to identify regions of the mouse GnRH receptor gene promoter that confer responsiveness to GnRH, PMA or cAMP. Also, we will determine if the mouse GnRH receptor gene contains high affinity binding site(s) for estrogen receptor. In Specific Aim 3, we propose to construct transgenic mice as an ultimate test for cell-specific expression of chimeric GnRH receptor genes and to serve as an in vivo model for studying hormonal regulation of GnRH receptor gene expression. Finally, limited progress has been made in identifying cis-acting elements or trans-acting factors required for GnRH activation of gonadotropin gene expression. Thus, we propose a fourth specific aim to address the molecular requirements for GnRH stimulation of expression of the glycoprotein hormone alpha subunit gene.