| Gonadotropin hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH), chorionic gonadotropin (hCG), and thyroid stimulating hormone (TSH) regulate diverse and essential reproductive functions and cellular metabolism. Gonadotropin action manifests with binding and activation of specific G-protein coupled receptors to induce transient accumulation of cyclic adenosine monophosphate (cAMP) and inositol phosphate (IP) from adenylyl cyclase and phospholipase C, respectively. The receptors possess two halves; extracellular hormone binding (exodomain) and signal transduction, membrane associated (endodomain) domains. The mechanism to induce two separate and distinct signals has not previously been addressed. Additionally, the regions within the endodomain responsible for signal origin, receptor activation, and signal transduction are undefined. It is clinically and scientifically advantageous to investigate the mechanisms of multiple signal generation because defining the individual signal importance would significantly improve upon our understanding of reproduction and clinical approaches toward reproductive dysfunction. Therefore, my research was designed and conducted to address specific questions about the origins, divergence and regulation of gonadotropin signals. Additionally, the relationship of the exodomain and the endodomain on hormone binding to the exodomain of the receptor was addressed.;Alanine scanning mutagenesis of the extracellular, adjoining eleven amino acid sequence linking transmembrane segments six and seven of the LH receptor (exoloop 3) was employed to investigate the origin of signal divergence. The mutant, Lys583 Ala, resulted in the complete loss of cAMP induction and further investigation by substitution with a panel of amino acids defined the lysine side chain as irreplaceable for cAMP induction but not IP induction. The results provide the first evidence of separate and distinct signal origins for cAMP and IP.;Additionally, the FSH receptor exoloop 3 was alanine scanned and tested for biological function. Unlike exoloop 3 mutants of the LH receptor, several alanine substitutions to the exoloop 3 of FSH receptor negatively impacted cAMP induction while improving hormone binding. Furthermore, FSH mutant receptors resulted in differential induction of individual inositol phosphates. This finding was most evident from multiple substitution for Lys590.;In conclusion, biological activities of gonadotropins are conducted through separate and distinct receptor mechanisms. Additionally, the endodomain constrains the exodomain and influences hormone binding. |
