GENERATION OF A NOVEL GNRHR CLONE TO STUDY RECEPTOR ACTIVITY

The hypothalamic-pituitary-gonadal axis is critical for reproductive function. The axis is controlled by the pulsatile release of gonadotropin releasing hormone (GnRH) from the hypothalamus. GnRH stimulates gonadotrope cells located in the anterior pituitary. This causes the concomitant pulsatile release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which act on the gonads to regulate reproductive processes. The GnRH receptor (GnRHR) is a unique G protein-coupled receptor that is distinguished by the lack of a carboxyl-terminal cytoplasmic tail. There are no suitable antibodies for the GnRHR, thus making the study of receptor modifications in response to ligand binding difficult. In this study we will create a cDNA clone of the mouse GnRHR with an extracellular tag encoding the V5 epitope derived from the SV5 paramyxovirus. The tagged gene will be expressed in LβT2 mouse gonadotrope cells and NIH/3T3 mouse fibroblast cells and monitored by fluorescent immunocytochemical analysis of transfected cells. Receptor function will be tested in transfected NIH/3T3 cells by monitoring activation of intracellular kinase signaling cascades in response to GnRH. We will examine the phosphorylation of the receptor in both LβT2 and NIH/3T3 cells in response to GnRH treatment using immunoprecipitation with anti-V5 antibody and Western blotting with anti-phosphoserine antibody. We will also examine the role of putative phosphorylation sites in receptor activation of signaling cascades and desensitization of activated receptor using site-directed mutagenesis.