The overall goal of this project is to gain an understanding at the molecular level of the mechanisms by which LH and prostaglandins act in the ovary. Currently, it centers more of LH than prostaglandins, specifically on the elucidation of structure- function relationships of the receptors for LH and FSH. Using as a base a newly developed cloning technique that involves the construction of genomic libraries in an eukaryotic cell line (mouse L cells) followed by functional screening of stably transfected cells for appearance of LH and FSH receptors as seen by their ability to stimulate adenylyl cyclase, and using an already cloned Gs-stimulating receptor as a model system, we proposed: 1. To clone the cDNAs for the LH and FSH receptors, deduce from them the predicted amino acid sequence, and explore their various functional domains as seen: 1) on caparison with other non-peptide hormone receptors that stimulate Gs. 2) on molecular engineering and construction of chimeras between these receptors and receptors with other hormonal and/or G protein specificity, so as to deduce the exact domains responsible for ligand interaction, G protein interaction, desensitization and down regulation (internalization). 2. To study the genomic organization of the LH and FSH receptor genes, and determine for the LH receptor the molecular basis for the different behavior of follicular and luteal LH receptors: is it due to existence of two (or more) LH receptor genes?, to different post-translational modifications?, or to a change in the quality of the Gs protein that couples the receptor to the effector function(s)? 3. To determine the biological importance of LB receptor desensitization and down-regulation for the luteinizing and luteolytic effects of LH as studied in transgenic mice that express LH receptors engineered to be desensitization- and/or down-regula- tion-negative. Likewise, we propose to determine the molecular basis for the resistance of FSH receptors to FSH-induced down regulation, and whether these receptors have activities other than stimulation of Gs, e.g., increased phosphoinositide turnover.
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