An novel gonadotrope cell line (LbetaT) has been recently developed which has many of the features of a mature gonadotrope including the expression of the GnRH receptor and both the glycoprotein alpha- subunit and the LH Beta-subunit proteins. We plan to use these cells to investigate GnRH signaling pathways. These experiments will involve the micro injection of antibodies and recombinant proteins into a single LbetaT cells, the use recombination adenoviruses, or cell-permeable inhibitor peptides to block or stimulate selective pathways. A number of end-points to monitor GnRH signaling will be used including LH secretion, activation of MAPKinase, induction of c- fos and jun-B, stimulation of BrdU incorporation, and inositol phosphate and diacylglygerol production.
Aim1. To test the hypotheses that Gq mediates GnRH signaling the LbetaT cells. We will determine whether the GnRH receptor couples to Gq. Signaling by the Galphaq subunit and Gbetagamma subunits will be blocked by specific inhibitors. Constitutively active versions will be introduced into cells to mimic GnRH stimulation.
Aim 2. To test the hypothesis that tyrosine phosphorylation of Gq associated proteins is required for GnRH signaling in LbetaT cells. We will investigate whether proteins associated with Gq are tyrosine phosphorylated and whether phosphorylation is required for GnRH signaling. A mutant Galphaq will be introduced into cells to determine whether signaling is impaired.
Aim 3. To test the hypothesis that GnRH -stimulated MAPKinase activation is ras dependent in LbetaT cells. We will investigate whether activation of MAPKinase by GnRH requires activation of ras. Is activated by GnRH. If ras independent, then we will determine whether PKC activates the pathway downstream of ras.
Aim 4. To test the hypothesis that GnRH utilizes multiple pathways to stimulate gene expression through distinct response elements in LbetaT cells. Reporter genes containing multimeric GnRH-response elements from the mouse and human alpha- and rat LH Beta-promoter will be constructed. These will be injected into LbetaT cells along with antibodies or recombinant proteins to block selective transcription factors or kinases.
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