The gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), are key regulators of ovarian and testicular function. The long term objectives of this project are to understand the structural and functional relationships between the gonadotropins and their specific cell surface receptors. This knowledge is important for rational design of hormone agonists and antagonists, and for diagnosis and treatment of infertility. In the ovary, LH acts through its receptor to regulate steroidogenesis, trigger ovulation, and promote formation of the corpus luteum. The proposed studies will focus on the structure/function relationships of the LH receptor (LHR) in the ovary. Our previous work has identified four binding determinants in the extracellular portion of the LH receptor. Studies with synthetic peptides and site-directed mutagenesis have identified two arginine residues in one determinant as being essential for proper receptor function. In the current grant, we plan to identify other amino acids that are critical for the functional activity of the LH receptor. Fluorescence anisotropy of synthetic receptor peptides will be used to assess specific associations with hormonal proteins, and amino acid substitutions will be employed to identify critical residues. Site- directed mutagenesis will be used to further evaluate and confirm the functional significance of residues in intact receptor. Mutated receptors will be analyzed in hormone binding and adenylyl cyclase assays. The LH receptor mRNA undergoes extensive alternate splicing, but the physiological relevance of this is unknown. Variant mRNA-s have not been well characterized in human ovary, and inappropriate splicing may be a cause of ovulatory dysfunction and luteal insufficiency. Polymerase chain reactions (PCR) and automated cDNA sequencing will be used to identify variant LHR mRNA's in human ovary and their characteristics will be correlated with phase of the menstrual cycle. Lastly, we have identified a natural mutation of the human LHR gene in a patient with ovulatory dysfunction and the mutation effects receptor activity. Genomic DNA from infertility patients will continue to be studied by PCR and direct sequencing to detect other mutations in the LHR gene. Mutations will be replicated in cloned LHR cDNA and transfection studies will be used to assess the functional activity of mutagenized receptors. These studies will increase our knowledge of the mechanisms involved in hormone-LH receptor interactions, transmembrane signalling, and the role of LHR in the pathogenesis of infertility.
